Sorting with counter selection using sequence similar peptides

ABSTRACT

The present invention relates to a method for selecting a cell or a virus expressing on its surface an antigen-binding protein specifically binding to a protein antigen of interest (PAI) while counter selection using a similar protein antigen (SPA) is applied. Further, the invention provides a method for determining the sequence of a nucleic acid encoding an antigen-binding protein or an antigen-binding part thereof and a method for producing a cell expressing a nucleic acid encoding an antigen-binding protein or an antigen-binding part thereof. The invention also relates to a method for treating a subject with a selected cell population.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/858,167, filed Jun. 6, 2019, and German Application No. 10 2019 129341.3, filed Oct. 30, 2019, the content of each of these applications isherein incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_3000058-017000_ST25.txt” createdon 4 Jun. 2020, and 31,154 bytes in size) is submitted concurrently withthe instant application, and the entire contents of the Sequence Listingare incorporated herein by reference.

The present invention relates to a method for selecting a cell or avirus expressing on its surface an antigen-binding protein specificallybinding to a protein antigen of interest (PAI) while counter selectionusing a similar protein antigen (SPA) is applied. Further, the inventionprovides a method for determining the sequence of a nucleic acidencoding an antigen-binding protein or an antigen-binding part thereofand a method for producing a cell expressing a nucleic acid encoding anantigen-binding protein or an antigen-binding part thereof. Theinvention also relates to a method for treating a subject with aselected cell population.

BACKGROUND OF THE INVENTION

The field of adoptive cell transfer (ACT) has become one of the mostpromising and innovative approaches to treat cancer, viral infectionsand other immune-modulated disease. To support the broader clinicalapplication of T-cell receptor (TCR)-modified T-cells, it is importantthat risks can be appropriately identified and mitigated, preferably atthe pre-clinical level. The toxicity observed to date with theadministration of TCR-modified T-cells is similar to that observedduring standard ACT and can be grossly divided into three main groups:toxicity due to the lymph depleting preparation regimen,cytokine-related toxicity and immune-related toxicity. Immune-relatedtoxicity can be classified into two subcategories: so-called“off-tumor/on-target” effects and “off-tumor/off-target” effects. Theoptimal gene-engineered T-cell therapy target antigen is one that isonly present on the tumor cell and absent in healthy cells; however, inmost cases the selected tumor target antigens are over-expressed oraberrantly expressed proteins that may be present to varying extent innormal cells (Johnson L A et al., Gene therapy with human and mouseT-cell receptors mediate cancer regression and targets normal tissuesexpressing cognate antigen, Blood 2009; 114:535-46).

“Off-Tumor/On-Target” Toxicity

Gene-engineered T-cell therapies may, therefore, trigger a potentcellular immune response against normal cells, even those that expressthe target antigens at low levels. This type of toxicity is known as“off-tumor/on-target” and is due to, for example, the engineered T-cellsbeing unable to distinguish between normal cells and cancer cells thatexpress the targeted antigen. Targeting of Melan A (MLA; also referredto as “melanoma antigen recognized by T-cells 1” (MART-1)) has beenassociated with significant “off-tumor/on-target” side effects (JohnsonL A et al., Gene therapy with and mouse T-cell receptors mediates cancerregression and targets normal tissue expressing cognate antigens, Blood2009, 114:535-46; van den Berg J H et al., Case report of a FatalSerious Adverse event upon Administration of T-cells transduced with aMART-1 specific T-cell Receptor, Mol. Ther. 2015; 23:1541-50).Specifically, a case report has been published describing a fatalserious adverse event 3 days after transduced T-cell administration witha MART-1 specific TCR to a patient with metastatic melanoma. InfusedT-cells were recovered from blood, broncho-alveolar lavage, ascites,tumor sites and heart tissue, and although no cross-reactivity of themodified T-cells toward a 3-D beating cardiomyocyte culture wasobserved, the authors were not able to exclude the possibility ofcross-reactivity with an allogeneic MHC-peptide complex. Additionally,multiple-organ failure was found to be due to on-target cytokinerelease. Off-tumor/on target toxicity can be avoided by selecting targetantigens that show a sufficiently low expression off-tumor to lead to anacceptable toxicity upon application of doses that are therapeuticallyeffective on the tumor.

“Off-Tumor/Off-Target” Toxicity

Because most tumor antigens are derived from self-proteins(tumor-associated antigens), the isolation of high-affinitytumor-specific T-cells is effectively precluded by thymic selection. TCRaffinity can, nevertheless, be considerably enhanced through mutation ofspecific regions within the complementarity-determining regions (CDRs).Although useful to promote modified T-cell efficacy, due to TCRdegeneracy, this approach carries the risk that a TCR might recognizeother related peptide antigens present on normal tissue throughcross-reactivity. Previously published results have shown lethaltoxicities in two patients, who were infused with T-cells engineered toexpress a TCR targeting melanoma-associated antigen A3 (MAGE-A3)cross-reacting with a peptide from the muscle protein Titin, even thoughno cross-reactivities had been predicted in the pre-clinical studies(Linette, G P et al., Cardiovascular toxicity and titin cross-reactivityof affinity enhanced T-cells in myeloma and melanoma, Blood 2013;122:863-71; Cameron, B J et al., Identification of a Titin-derivedHLA-A1-presented peptide as a cross-reactive target for engineeredMAGE-A3 directed T-cells, Sci. Transl. Med. 2013; 5:197-103). Thesepatients demonstrated that TCR-engineered T-cells can have serious andnot readily predictable off-target and organ-specific toxicities andhighlight the need for improved methods to define the specificity ofengineered TCRs. Strategies such as peptide scanning and the use of morecomplex cell structures are therefore recommended in pre-clinicalstudies to mitigate the risk of off-target toxicities in future clinicalinvestigations. Therefore, there is still an unmet medical need todevelop and provide TCRs with low off-tumor/off-target toxicity. Thepresent invention provides methods to rapidly identify antigen bindingmolecules, in particular TCRs that specifically and selectively bind totheir target antigens and, thus provide enhanced safety profiles andreduced cross-reactivity to sequence similar target antigens, inparticular sequence similar peptides on healthy tissues. The rapid,preferably one step, selection method of the present invention isparticularly useful in the identification of patient-derived T-cellsexpressing TCRs with desired anti-tumor activity.

SUMMARY OF THE INVENTION

A first aspect of the invention relates to a method for selecting a cellor a virus expressing on its surface an antigen-binding proteinspecifically and/or selectively binding to a protein antigen of interest(PAI) comprising the following steps:

-   (i) providing a cell population comprising cells or a virus    population;-   (ii) contacting the cell population or the virus population of    step (i) with a first antigen complex (1^(st) AC) comprising the PAI    and a detectable label A or with the PAI comprising a detectable    label A;-   (iii) contacting the cell population or the virus population of    step (i) with at least a second antigen complex (2^(nd) AC)    comprising a similar protein antigen (SPA), wherein the amino acid    sequence of the SPA differs by at least 1 amino acid from the amino    acid sequence of the PAI and wherein the 2^(nd) AC comprises a    detectable label B; or with the SPA and a detectable label B; and-   (iv) selecting at least one cell or virus that specifically and/or    selectively binds to the 1^(st) AC,    wherein the detectable label A and the detectable label B are    detectably different from each other.

A second aspect of the invention further relates to a method fordetermining the sequence of a nucleic acid encoding an antigen-bindingprotein or an antigen-binding part thereof comprising the steps of:

-   (i) isolating the nucleic acid encoding the antigen-binding protein    or the antigen-binding part thereof from the cell selected in the    method of the first aspect of the invention; and-   (ii) determining the sequence of the nucleic acid.

A third aspect of the invention relates to a method for producing a cellexpressing a nucleic acid encoding an antigen-binding protein or anantigen-binding part thereof comprising the steps of:

-   (i) providing the nucleic acid sequence encoding the antigen-binding    protein or an antigen-binding part thereof from the cell selected in    the method of the first aspect of the invention;-   (ii) producing a nucleic acid vector comprising the nucleic acid    sequence provided in step (i) optionally under the control of an    expression control element; and-   (iii) introducing the nucleic acid vector of step (ii) into a host    cell.

A fourth aspect of the invention relates to a method for treating asubject in need thereof comprising the steps of:

-   (i) providing a cell population of the subject comprising immune    cells;-   (ii) contacting the cell population of step (i) with a first antigen    complex (1^(st) AC) comprising a PAI and a detectable label A;-   (iii) contacting the cell population of step (i) with at least a    second antigen complex (2^(nd) AC) comprising a SPA, wherein the    amino acid sequence of the SPA differs by at least 1 amino acid from    the amino acid sequence of the PAI and wherein the 2^(nd) AC    comprises a detectable label B; and-   (iv) selecting at least one cell that specifically binds to the    1^(st) AC,    wherein the detectable label A and the detectable label B are    detectably different from each other-   (v) increasing the number of the at least one selected cell selected    in step (iv) by cultivation; and-   (vi) reintroducing the cultivated cells into the subject.

A fifth aspect of the invention relates to a method for selecting animmune cell expressing on its surface an antigen-binding proteinspecifically binding to a protein antigen of interest (PAI) comprisingthe following steps:

-   (i) providing a cell population comprising immune cells;-   (ii) contacting the cell population of step (i) with a first antigen    complex (1^(st) AC) comprising the PAI and a detectable label A or    with the PAI comprising a detectable label A;-   (iii) contacting the cell population of step (i) with at least a    second antigen complex (2^(nd) AC) comprising an irrelevant protein    antigen (IPA), wherein the amino acid sequence of the IPA when    aligned with the amino acid sequence of the PAI is identical to the    PAI at two amino acids positions or less and wherein the IAC    comprises a detectable label G; or with the IPA and a detectable    label G; and-   (iv) selecting at least one cell that specifically binds to the    1^(st) AC,    wherein the detectable label A and the detectable label G are    detectably different from each other.

LIST OF FIGURES

In the following, the content of the Figures comprised in thisspecification is described. In this context please also refer to thedetailed description of the invention above and/or below.

FIG. 1 shows a schematic presentation of two exemplifying applicationsof the invention. The upper pathway of the figure represents the use ofthe gating strategy when applied to primed T-cells that underwent anindividual T cell culturing step, the lower part of the figure shows theuse of the gating strategy in a direct sorting approach, wherein aheterogenous T cell population obtained from a natural repertoire isenriched with target specific T cells using the gating strategy. In bothexamples the positively sorted cell fraction represents immune cellsexpressing on their surface an antigen-binding protein specificallyand/or selectively binding to a protein antigen of interest.Abbreviations used in the figure: SPA: similar protein antigen, PAI:protein antigen of interest, APC: antigen-presenting cell.

FIG. 2 shows an exemplary gating strategy of non-amplifiedtarget-specific T cells. To enhance the frequency of low-frequencytarget-specific T cells in the test sample, the cells have been enrichedby fluorochrome-tetramer specific magnetic bead isolation. Subsequently,cells were stained for surface markers and assessed by flow cytometry.Individual 2D-color tetramer combinations were used to staintarget-specific and similar peptide-specific T cells. In this example1.65% of CD8 T cells bind to target-peptide tetramer and of thosetarget-specific CD8 T cells 29.4% also bind to similar peptide-tetramer(Target⁺/SIM⁺), which is comprised of 3 different similar peptide-HLAs.By including similar peptide tetramers in the sorting procedure, a highproportion of cross-reactive T cells (Target⁺/SIM⁺) can be excluded.

FIG. 3 shows an exemplary gating strategy of primed T cell populations.Individual T cell cultures were repeatedly stimulated with targetpeptide HLA-coated artificial presenting cells to enhance low-frequencytarget-specific CD8 T cells. After 4 weeks in culture those primed Tcell cultures were stained for surface markers and individual 2D-colortetramer combinations for target-HLA and 3 similar peptide-HLAs. Theupper panel shows a monoclonally enriched T cell population binding toboth target- and similar-peptide tetramers (Target⁺/SIM⁺). The lowerpanel shows a monoclonally enriched T cell population binding only tothe target- but not similar-peptide tetramer. By including similarpeptide tetramers in the staining procedure cross-reactive T cells(Target⁺/SIM⁺) can be excluded from sorting.

FIG. 4 shows that TCRs from T cells sorted using target-peptidetetramers only, can be cross-reactive to target-similar peptides. TCRsidentified using target-peptide tetramers were assessed forcross-reactivity against 10 target similar peptides after mRNAelectroporation into healthy donor T cells. As measure for reactivity,IFNγ secretion upon co-culture with peptide-loaded T2 cells wasassessed. All TCRs in this example react against the target peptide(positive control) and not against controls, which areunrelated/irrelevant peptide loaded T2 cells, unloaded T2 cells oreffector only cells. However, the TCRs in FIG. 4A and FIG. 4B also showreactivity to similar peptides, namely similar peptide 1 and 10 for TCRin FIG. 4A and similar peptide 9 and 10 for the TCR in FIG. 4B. Only theTCR in FIG. 4C shows no cross-reactivity and is therefore selected forfurther characterization.

FIG. 5 shows the functional assessment of a TCR isolated from T cellsbinding to target-peptide tetramers only (TCR PAI+/SPA−), as well as acontrol TCR specific for a control peptide (“control peptide”), and a noTCR control (“no peptide”). For this end, TCR-mRNA was electroporatedinto NFAT-luciferase Jurkat reporter cells and their activation assessedafter co-culture with peptide/target similar peptides (“SIM 1, SIM 2,and SIM 3”) loaded T2 antigen-presenting cells. The TCR derived fromPAI+/SPA− sorted T cells triggers activation only when co-cultured withtarget peptide-loaded T2 cells. The control TCRs shows reactivity in thepresence of control-peptide and Jurkat cells without TCR mRNAelectroporation do not respond to peptide-loaded T2 cells. This exampleshows that TCRs binding to target-peptide tetramers also show reactivitytoward those peptides on a functional level.

FIGS. 6, 7 and 8 show peptide presentation profiles of a target similarpeptide 1 (TSP1) (FIG. 6), TSP2 (FIG. 7) and an irrelevant peptide (IP;FIG. 8) from Example 4 based on XPRESIDENT mass spectrometry data. Upperpart: Median relative MS signal intensities from technical replicatemeasurements are plotted as colored dots for single HLA-A*02 normalsamples on which the peptide was detected. Normal samples are groupedaccording to organ of origin. Box-and-whisker plots represent normalizedsignal intensities over multiple samples and have been defined in thelog space. Boxes display median, 25^(th) and 75^(th) percentile.Whiskers extend to the lowest data point still within 1.5 interquartilerange (IQR) of the lower quartile, and the highest data point stillwithin 1.5 IQR of the upper quartile. Lower part: The peptide detectionfrequency in every organ is shown as a bar plot. Numbers below the panelindicate number of samples on which the peptide was detected out of thetotal number of samples analyzed for each organ (N≥628 for normalsamples across all organs). If the peptide has been detected on a samplebut could not be quantified for technical reasons, the sample isincluded in this representation of detection frequency, but no dot isshown in the upper part of the figure. adipose: adipose tissue; adrenalgl: adrenal gland; bladder: urinary bladder; bloodvess: blood vessel;esoph: esophagus; gall bl:gallbladder; intest. la: large intestine;intest. sm: small intestine; nerve cent: central nerve; nerve periph:peripheral nerve; parathyr: parathyroid gland; petit: peritoneum;pituit: pituitary; skel. mus: skeletal muscle.

LIST OF SELECTED SEQUENCES

-   SEQ ID NO: 1 X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈, wherein X₁-X₈ are amino acids    positions in a target peptide of a length of 8 amino acids and X in    each case is any amino acid;-   SEQ ID NO: 2 X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉, wherein X₁-X₉ are amino    acids positions in a target peptide of a length of 9 amino acids and    X in each case is any amino acid;-   SEQ ID NO: 3 X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀, wherein X₁-X₁₀ are    amino acids positions in a target peptide of a length of 10 amino    acids and X in each case is any amino acid;-   SEQ ID NO: 4 X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁, wherein X₁-X₁₁ are    amino acids positions in a target peptide of a length of 11 amino    acids and X in each case is any amino acid;-   SEQ ID NO: 5 X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂, wherein X₁-X₁₂    are amino acids positions in a target peptide of a length of 12    amino acids and X in each case is any amino acid.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tolimit the scope of the present invention which will be limited only bythe appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions etc.), whether supra or infra, is hereby incorporated byreference in its entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention. Some of the documents cited herein arecharacterized as being “incorporated by reference”. In the event of aconflict between the definitions or teachings of such incorporatedreferences and definitions or teachings recited in the presentspecification, the text of the present specification takes precedence.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Definitions

To practice the present invention, unless otherwise indicated,conventional methods of chemistry, biochemistry, and recombinant DNAtechniques are employed which are explained in the literature in thefield (cf., e.g., Molecular Cloning: A Laboratory Manual, 2^(nd)Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press,Cold Spring Harbor 1989).

In the following, some definitions of terms frequently used in thisspecification to characterize the invention are provided. These termswill, in each instance of its use, in the remainder of the specificationhave the respectively defined meaning and preferred meanings.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents, unless the contentclearly dictates otherwise.

The term “amino acid” refers in the context of this invention to anymonomer unit that comprises a substituted or unsubstituted amino group,a substituted or unsubstituted carboxy group, and one or more sidechains or groups, or analog of any of these groups. Exemplary sidechains include, e.g., thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto,azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynl,ether, borate, boronate, phospho, phosphono, phosphine, heterocyclic,enone, imine, aldehyde, ester, thioacid, hydroxylamine, or anycombination of these groups. Other representative amino acids include,but are not limited to, amino acids comprising photoactivatablecross-linkers, metal binding amino acids, spin-labelled amino acids,fluorescent amino acids, metal-containing amino acids, amino acids withnovel functional groups, amino acids that covalently or noncovalentlyinteract with other molecules, photocaged and/or photoisomerizable aminoacids, radioactive amino acids, amino acids comprising biotin or abiotin analog, glycosylated amino acids, other carbohydrate modifiedamino acids, amino acids comprising polyethylene glycol or polyether,heavy atom substituted amino acids, chemically cleavable and/orphotocleavable amino acids, carbon-linked sugar-containing amino acids,redox-active amino acids, amino thioacid containing amino acids, andamino acids comprising one or more toxic moieties. As used herein, theterm “amino acid” includes the following twenty natural or geneticallyencoded alpha-amino acids: alanine (Ala or A), arginine (Arg or R),asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C),glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G),histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine(Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline(Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp orW), tyrosine (Tyr or Y), and valine (Val or V). In cases where “X”residues are undefined, these are to be interpreted as “any amino acid.”The structures of these twenty natural amino acids are shown in, e.g.,Stryer et al., Biochemistry, 5th ed., Freeman and Company (2002).Additional amino acids, such as selenocysteine and pyrrolysine, can alsobe genetically coded for (Stadtman (1996) “Selenocysteine,” Annu RevBiochem. 65:83-100 and Ibba et al. (2002) “Genetic code: introducingpyrrolysine,” Curr Biol. 12(13):R464-R466). The term “amino acid” alsoincludes unnatural amino acids, modified amino acids (e.g., havingmodified side chains and/or backbones), and amino acid analogs. See,e.g., Zhang et al. (2004) “Selective incorporation of5-hydroxytryptophan into proteins in mammalian cells,” Proc. Natl. Acad.Sci. U.S.A. 101(24):8882-8887, Anderson et al. (2004) “An expandedgenetic code with a functional quadruplet codon” Proc. Natl. Acad. Sci.U.S.A. 101(20):7566-7571, Ikeda et al. (2003) “Synthesis of a novelhistidine analogue and its efficient incorporation into a protein invivo,” Protein Eng. Des. Sel. 16(9):699-706, Chin et al. (2003) “AnExpanded Eukaryotic Genetic Code,” Science 301(5635):964-967, James etal. (2001) “Kinetic characterization of ribonuclease S mutantscontaining photoisomerizable phenylazophenylalanine residues,” ProteinEng. Des. Sel. 14(12):983-991, Kohrer et al. (2001) “Import of amber andochre suppressor tRNAs into mammalian cells: A general approach tosite-specific insertion of amino acid analogues into proteins,” Proc.Natl. Acad. Sci. U.S.A. 98(25):14310-14315, Bacher et al. (2001)“Selection and Characterization of Escherichia coli Variants Capable ofGrowth on an Otherwise Toxic Tryptophan Analogue,” J. Bacteriol.183(18):5414-5425, Hamano-Takaku et al. (2000) “A Mutant Escherichiacoli Tyrosyl-tRNA Synthetase Utilizes the Unnatural Amino AcidAzatyrosine More Efficiently than Tyrosine,” J. Biol. Chem.275(51):40324-40328, and Budisa et al. (2001) “Proteins with{beta}-(thienopyrrolyl) alanines as alternative chromophores andpharmaceutically active amino acids,” Protein Sci. 10(7):1281-1292.Amino acids can be merged into peptides, polypeptides, or proteins. Asused in this specification the term “peptide” refers to a short polymerof amino acids linked by peptide bonds. It has the same chemical(peptide) bonds as proteins but is commonly shorter in length. Theshortest peptide is a dipeptide, consisting of two amino acids joined bya single peptide bond. There can also be a tripeptide, tetrapeptide,pentapeptide, etc. Typically, a peptide has a length of up to 8, 10, 12,15, 18 or 20 amino acids. A peptide has an amino end and a carboxyl end,unless it is a cyclic peptide.

The term “virus” refers in the context of the present invention to smallobligate intracellular parasites, which by definition contain either aRNA or DNA genome surrounded by a protective protein coat, i.e. acapsid. The genome of a virus may consist of DNA or RNA, which may besingle stranded (ss) or double stranded (ds), linear or circular. Theentire genome may occupy either one nucleic acid molecule (monopartitegenome) or several nucleic acid segments (multipartite genome). Thevirus can be a double-stranded DNA virus, preferably Myoviridae,Siphoviridae, Podoviridae, Herpesviridae, Adenoviridae, Baculoviridae,Papillomaviridae, Polydnaviridae, Polyomaviridae, Poxviridae; asingle-stranded DNA virus, preferably Anelloviridae, Inoviridae,Parvoviridae; double-stranded RNA virus, preferably Reoviridae; asingle-stranded RNA virus, preferably Coronaviridae, Picornaviridae,Caliciviridae, Togaviridae, Flaviviridae, Astroviridae, Arteriviridae,Hepeviridae; negative-sense single-stranded RNA virus, preferablyArenaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Bunyaviridae,Orthomyxoviridae, Bornaviridae; a single-stranded RNA reversetranscribing virus, preferably Retroviridae; or a double-stranded RNAreverse transcribing virus, preferably Caulimoviridae, Hepadnaviridae.

The term “bacteriophage” (or “phage”) refers in the context of thepresent invention to a virus that infects and replicates in bacteria andarchaea. Bacteriophages are dependent on a host organism, typicallybacteria, to replicate in and inject their genome, which is eithercomprised of proteins that encapsulate desoxyribonucleic acid (DNA) orribonucleic acid (RNA), into the host organims's cytoplasm. Prominentexamples of bacteriophages used in biotechnology are bacteriophage T4lambda (T4λ) phage, T7 phage, fd filamentous phage, in particularfilamentous M13 phage of which all have certain benefits and drawbacks.

The term “virus population” refers in the context of the presentinvention to a high number of viruses which differ in the geneticinformation encoding the antigen binding protein expressed on theirsurface. The viral population can thus, express a library ofheterologous antigen binding proteins.

The term “phage display” or “phage library” refers in the context of thepresent invention to a system that is used for high-throughput screeningof protein interactions. Briefly, a gene encoding a protein of interestis inserted into a bacteriophage coat protein gene which causes thebacteriophage to “display”, i.e. to show, the protein on its surfacewhile keeping the gene encoding the protein of interest in its DNA orRNA. This results in a connection of genotype and phenotype. Theproteins which are displayed on the bacteriophage's surface cansubsequently be screened against other proteins, peptides or DNAsequences to study their interaction between the displayed molecule andthe molecules to be screened. Such a molecule can be an antibody or afragment thereof, a TCR or a fragment thereof, a BCR or a fragmentthereof or a CAR or a fragment thereof. Fragments of TCRs may comprisethe alpha variable domain and the beta variable domain. Fragments in thecontext of the present inventions are also defined in detail below.

The term “cell” refers in the context of the present invention toeukaryotic cells which contain a nucleus and cell organelles and can befound in protozoa, fungi, plants and animals. Animals can comprisemammalian cells. Mammalian cells comprise inter alia human cells,rhodent cells, such as mouse, or rat cells, monkey cells, pig cells ordog cells. Fungi cells inter alia comprise yeast cells. Typical yeastcells used in biotechnology, for example in a yeast surface display, areSaccharomyces cerevisiae cells.

The term “yeast surface display” or “yeast display” or “yeast library”refers in the context of the present invention to a protein engineeringtechnique using yeast cells that express recombinant proteins ofinterest and incorporate these proteins into their cell wall. Thisallows for isolation and engineering of proteins, in particularantibodies or fragments thereof, TCRs or fragments thereof, BCRs orfragments thereof or CARs or fragments thereof. In detail, in the yeastsurface display, the unit of selection is a yeast cell that is decoratedwith tens of thousands of copies of the protein of interest and thatcarries the plasmid encoding that protein. The plasmid can be shuttledbetween Saccharomyces cerevisiae, for display and sorting, and E. coli,for DNA preparation and molecular biology. In the form of yeast displaypioneered by the Wittrup group (Chao et al., 2006), each ¹⁰Fn3 variantis expressed as a genetic fusion with a native yeast protein found inthe cell wall, Aga2p. Aga2p is a domain of the native yeast, anagglutinin mating factor; typically, it is cloned upstream of thesequence encoding the ¹⁰Fn3 variant. In addition, an epitope tag, suchas c-myc and V5, is engineered immediately downstream from the sequenceencoding the ¹⁰Fn3 variant. Upon induction, the mating-factor secretorysignal peptide directs the fusion protein to be exported from the cell;it is captured on the surface of the yeast cell wall by its bindingpartner, Aga1p, to which it forms two disulfide bonds. The result is aculture where each yeast cell displays between 10,000 and 100,000 copiesof a single ¹⁰Fn3 variant. On average, the more thermostable thevariant, the larger the number of its molecules on the yeast surface(Hackel et al., 2010).

The term “immune cell” refers in the context of this invention to a cellof the immune system. The immune system comprises different cell typessuch as precursor cells comprising lymphoid stem cells, which ultimatelydifferentiate into B and T lymphocytes and natural killer (NK) cells,and myeloblasts, which ultimately differentiate into granulocytes andmonocytes as well as fully differentiated leukocytes. Differentiatedleukocytes are thymus-, spleen-, bone marrow or lymph node-derived cellsand can be categorized into the main groups of granulocytes,B-lymphocytes, T-lymphocytes and monocytes, macrophages, and mast cellsand dendritic cells. Granulocytes are further divided into neutrophil,eosinophil and basophil granulocytes, which phagocytose bacteria, virusor fungi in the blood circulation. B-lymphocytes are precursors ofplasma cells and B-memory cells. The group of T-cells comprisesregulatory T-cells, memory T-cells, T helper cells and cytotoxicT-cells. While T helper cells activate plasma cells and natural killercells, regulatory T-cells inhibit the function of B and other T-cellsand thus, slow down the immune response. T memory cells are long-livingand possess a memory for specific antigens, and cytotoxic T-cellsrecognize and kill tumor cells or cells attacked by viruses byinteracting with tumor antigens or antigens of the attacked cells.Examples of T-cells and their surface phenotype described by thespecific surface markers of the respective T-cells are given in belowTable 1 (according to Dong and Martinez, Nature Reviews Immunology,2010):

TABLE 1 Common T-cell surface markers (non-exhaustive enumeration).Cell: Surface marker: Cytotoxic T-cells: αβ TCR, CD3, CD8 RegulatoryT-cells: αβ TCR, CD3, CD4 Regulatory T-cells αβ TCR, CD3, CD4, CD25,CTLA4, GITR (natural and inducible): Natural Killer cells: NK1.1,SLAMF1, SLAMF6, TGFβ, Vα24, Jα18 T helper cells: TH1 cells αβ TCR, CD3,CD4, IL-12R, IFNγR, CXCR3; TH2 cells αβ TCR, CD3, CD4, IL-4R,IL33R,CCR4, IL-17RB, CRTH2; TH9 cells αβ TCR, CD3, CD4; TH17 cells αβ TCR,CD3, CD4, IL-23R, CCR6, IL-1R, CD161; TH22 cells αβ TCR, CD3, CD4,CCR10; TILs Tumor-infiltrating lymphocytes T memory cells CCR7 hi, CD44,CD62Lhi, TCR, CD3, IL-7R (CD127), IL-15R

The term “tumor-infiltrating lymphocytes” (TILs) refers in the contextof the present invention to T-cells and B-cells that have migratedtowards a tumor and can often be found in the tumor stroma or the tumoritself. TILs typically comprise a cell population of white blood cellsthat may be used in ACT or autologous cell therapy. Such therapies havealready shown promising results, for example in patients with metastaticmelanoma in a variety of clinical trials (Guo et al.; “Recent updates oncancer immunotherapy”; Precision Clinical Medicine, 1(2), 2018-65-74).In the context of ACT, TILs are expanded ex vivo from surgicallyresected tumors or single cell suspensions isolated from tumorfragments. TILs are expanded with a high doses of cytokines, for exampleIL-2. Selected TIL lines that presented best tumor reactivity are thenfurther expanded in a “rapid expansion protocol” (REP), which usesanti-CD3 activation for a typical period of two weeks. The finalpost-REP TIL is infused back into the patient. The process can alsoinvolve a preliminary chemotherapy regimen to deplete endogenouslymphocytes in order to provide the adoptively transferred TILs withenough access to surround the tumor sites.

The term “immune cell enriched fraction” refers in the context of thisinvention to a cell population, which is derived from a naturallyoccurring cell population, e.g. blood, in which the relative abundanceof the immune cells has been increased in comparison to their abundancein the naturally occurring cell mixture. One ml of blood of a healthyhuman subject comprises, e.g. 4.7 to 6.1 million (male), 4.2 to 5.4million (female) erythrocytes, 4,000-11,000 leukocytes and200,000-500,000 thrombocytes. Thus, in blood immune cells onlyconstitute 0.06% to 0.25% of the total number of blood cells. An immunecell enriched fraction of blood thus may comprise more than 0.25%, morepreferably more than 10%, even more preferably more than 50%, even morepreferably more than 80% and most preferably more than 90% immune cells.The immune cell enriched fraction may be enriched for one or moresubtypes of immune cells. For example, the immune cell enriched fractionmay be enriched for lymphoid stem cells, T-cells, B-cells, plasma cellsor combinations. Usually, immune cells in immune cell enriched fractionsare selected by using one or more fluorescently labelled antibodies thatspecifically bind to a surface marker of the immune cells of interest.Suitable surface markers to select T-cells or sub-fractions within thegroup of T-cells are indicated in table 1 above. Cytotoxic T-cells canbe selected, e.g. by using an antibody that specifically binds to CD8 orby using antibodies that specifically bind to CD8 and CD3.

The term “cell population” refers in the context of this invention to aplurality of cells which may be homogenous or heterogenous, i.e. amixture of cells of different characteristic. Blood is an example of acell population which is a mixture of different cells. Homogenous cellpopulations can be obtained by selection of a particular subtype or byclonal expansion.

The term “antigen binding protein” refers in the context of thisinvention to one polypeptide or a complex of two or more polypeptidesthat comprise a paratope (alternatively referred to as “antigen bindingsite”) that specifically binds to an antigen. Examples of antigenbinding proteins are single chain antibodies, single chain TCRs,chimeric antigen receptor (CAR) and examples of antigen bindingcomplexes are antibodies, B cell receptors (BCRs) or TCRs.

The term “chimeric antigen receptor” (CAR; also known as chimericimmunoreceptor, chimeric T cell receptor, artificial T cell receptor) inthe context of the present invention refers to engineered receptors,which graft an arbitrary specificity onto an immune effector cell,preferably a T cell. Cells are genetically equipped with a CAR, which isa composite membrane receptor molecule and provides both targetingspecificity and T cell activation. The most common form of CARs arefusions of single chain variable fragment (scFv) derived from monoclonalantibodies, fused to CD3 transmembrane- and endodomain. The CAR targetsthe T cell to a desired cellular target through an antibody-derivedbinding domain in the extracellular moiety, and T cell activation occursvia the intracellular moiety signalling domains when the target isencountered. The transfer of the coding sequence of these receptors intosuitable cells, in particular T cells, is commonly facilitated by retro-or lentiviral vectors. The receptors are called chimeric because theyare composed of parts from different sources.

The term “epitope” refers in the context of this invention to thefunctional epitope of an antigen. The functional epitope comprises thoseresidues, typically amino acids or polysaccharides that contribute tothe non-covalent interaction between the paratope of the antigen bindingprotein and the antigen. The non-covalent interaction compriseselectrostatic forces, van der Walls forces, hydrogen bonds, andhydrophobic interaction. The functional epitope is a subgroup of theresidues that constitute the structural epitope of an antigen bindingprotein. The structural epitope comprises all residues that are coveredby an antigen binding protein, i.e. the footprint of an antigen bindingprotein. Typically, the functional epitope of an antigen bound by anantibody comprises 4 to 10 amino acids. Similarly, the functionalepitope of a peptide that is MHC presented typically comprises 4 to 8amino acids.

The term “expression” refers in the context of this invention to thepresence of a protein or peptide, in particular a PAI or a SPA in humantissue. The term expression of a protein or peptide means that it istranslated from its nucleic acid sequence into its amino acid sequenceduring the process of protein biosynthesis in the ribosomal machinery ofthe cell. The expressed protein can be located intracellularly orextracellularly, e.g. on the surface of cell. The human tissue whereinthe protein is expressed may be healthy or diseased tissue.

The term “protein antigen of interest” (PAI) refers in the context ofthis invention to a protein or a portion of a protein or a proteincomplex that comprises an epitope that is specifically bound by theparatope of an antigen-binding protein. A PAI is typically a naturallyoccurring protein and can be of any length. It is preferred that the PAIcomprises at least 25 amino acids. If that the PAI is specifically boundby a TCR accordingly, it is preferred that the length of the PAI is 8 to12 amino acids. The PAI may be a tumor associated target antigen (TAA),a viral protein or a bacterial protein. The PAI is typically a tumorassociated antigen (TAA), which is to be specifically targeted in, e.g.a tumor therapy.

The term “humanized mice” refers in the context of the present inventionto genetically modified mice which carry human genes, cells, tissuesand/or organs that exert their biological function, e.g. are intactregarding their biological function. Typically, immunodeficient mice areused as recipients for human cells or tissues, because they canrelatively easily accept heterologous cells or tissues due to lack ofhost immunity. Examples of humanized mice are the nude mouse, the severecombined immunodeficiency (SCID) mouse, the NCG mouse, the NOG(NOD/Shi-scid/IL-2Rγ^(null)) mouse or the NSG (NOD scid gamma) mouse.Mice that accept human version of genes into their respective mouse lociare called “knock-in” mice. B-cells and T-cells can be isolated fromhumanized mice and be used in the methods of the present invention.

The term “T-cell receptor libraries” refers in the context of thepresent invention to a library that contains a high number of differentT cell receptor (TCR) proteins or fragments thereof, wherein each TCRprotein or fragment thereof is different.

A “viral antigenic peptide” in the context of the present invention isshorter fragment of a viral protein that is presented by a majorhistocompatibility complex (MHC) molecule on the surface of an antigenpresenting cell, which is typically a diseased cell. The viral antigenicpeptide is of a viral origin, i.e. the cell is typically infected bysaid virus. The viral antigenic peptide in the context of the presentinvention may be an antigenic peptide selected from the group consistingof human immune deficiency virus (HIV) antigenic peptides, humancytomegalovirus (HCMV) antigenic peptides, cytomegalovirus (CMV)antigenic peptides, human papillomavirus (HPV) antigenic peptides,hepatitis B virus (HBV) antigenic peptides; hepatitis C virus (HCV)antigenic peptides; Epstein-Barr virus (EBV) antigenic peptides,Influenza antigenic peptides, preferably HIV, HBV, Influenza and HCMVantigenic peptides. Viral antigenic peptides can be used in the methodand the embodiments described herein include, for example, viralantigenic peptides as described in table 2 below. In one aspect, viralantigenic peptides that are used in the method and embodiment describedherein include at least one viral antigenic peptide comprising orconsisting of an amino acid sequence selected from the amino acidsequences of SEQ ID NO: 6 to SEQ ID NO: 8.

TABLE 2 List of viral antigenic peptides SEQ ID Amino acid NO: sequenceVirus MHC 6 SLYNTVATL HIV HLA-A*02:01 7 GILGFVFTL Influenza AHLA-A*02:01 8 NLVPMVATV HCMV HLA-A*02:01

A “bacterial antigenic peptide” in the context of the present inventionis shorter fragment of a bacterial protein that is presented by an MHCmolecule on the surface of an antigen presenting cell, which istypically a diseased cell. The bacterial antigenic peptide is of abacterial origin, i.e. the cell is typically infected by a bacterium.Such bacterial antigenic peptides have been discovered in the context ofinfections from, for example, Mycobacterium tuberculosis. Accordingly,the bacterial antigenic peptide in the context of the present inventionmay be a Mycobacterium tuberculosis antigenic peptide.

The term “tumor associated antigen” (TAA) refers in the context of thisinvention to autologous cellular antigens derived from all proteinclasses, such as enzymes, receptors, transcription factors, etc. thatare preferentially or exclusively expressed by tumor cells. TAA can bebroadly categorized into aberrantly expressed self-antigens, mutatedself-antigens, and tumor-specific antigens. TAAs that are preferentiallyexpressed by tumor cells, are also found in normal tissues. However,their expression differs from that of normal tissues by their degree ofexpression in the tumor, by alterations in their protein structure incomparison with their normal counterparts, or by their aberrantsubcellular localization within tumor cells. The TAA peptides that canbe used in the methods and embodiments described herein include, forexample, TAA peptides described in U.S. Publication 20160187351, U.S.Publication 20170165335, U.S. Publication 20170035807, U.S. Publication20160280759, U.S. Publication 20160287687, U.S. Publication 20160346371,U.S. Publication 20160368965, U.S. Publication 20170022251, U.S.Publication 20170002055, U.S. Publication 20170029486, U.S. Publication20170037089, U.S. Publication 20170136108, U.S. Publication 20170101473,U.S. Publication 20170096461, U.S. Publication 20170165337, U.S.Publication 20170189505, U.S. Publication 20170173132, U.S. Publication20170296640, U.S. Publication 20170253633, U.S. Publication 20170260249,U.S. Publication 20180051080, and U.S. Publication No. 20180164315, thecontents of each of these publications and sequence listings describedtherein, which are herein incorporated by reference in their entirety.Furthermore, the TAA in the context of the present invention is aspecific ligand of MHC-class-I-molecules or MHC-class-II-molecules,preferably MHC-class-I-molecules.

In an aspect, the antigen binding protein selected by the method of thepresent invention selectively recognize cells which present a TAApeptide described in one of more of the patents and publications listedabove. In another aspect, TAA peptides that may be used in the methodsand embodiments described herein include at least one TAA consisting ofan amino acid sequence selected from the amino acid sequences of SEQ IDNO: 9 to 164. In an aspect, the antigen binding protein selected by themethod of the present invention selectively binds cells which present aTAA peptide/MHC complex, wherein the TAA peptide comprises or consistsof an amino acid sequence of SEQ ID NO: 1 to 164. Further examples ofTAAs are listed in table 3.

TABLE 3 List of TAAs SEQ ID Amino Acid NO: Sequence 9 YLYDSETKNA 10HLMDQPLSV 11 GLLKKINSV 12 FLVDGSSAL 13 FLFDGSANLV 14 FLYKIIDEL 15FILDSAETTTL 16 SVDVSPPKV 17 VADKIHSV 18 IVDDLTINL 19 GLLEELVTV 20TLDGAAVNQV 21 SVLEKEIYSI 22 LLDPKTIFL 23 YLMDDFSSL 24 KVWSDVTPL 25LLWGHPRVALA 26 KIWEELSVLEV 27 LLIPFTIFM 28 FLIENLLAA 29 LLWGHPRVALA 30FLLEREQLL 31 SLAETIFIV 32 TLLEGISRA 33 ILQDGQFLV 34 VIFEGEPMYL 35SLFESLEYL 36 SLLNQPKAV 37 GLAEFQENV 38 KLLAVIHEL 39 TLHDQVHLL 40TLYNPERTITV 41 KLQEKIQEL 42 SVLEKEIYSI 43 RVIDDSLVVGV 44 VLFGELPAL 45GLVDIMVHL 46 FLNAIETAL 47 ALLQALMEL 48 ALSSSQAEV 49 SLITGQDLLSV 50QLIEKNWLL 51 LLDPKTIFL 52 RLHDENILL 53 GLPSATTTV 54 GLLPSAESIKL 55KTASINQNV 56 SLLQHLIGL 57 YLMDDFSSL 58 LMYPYIYHV 59 KVWSDVTPL 60LLWGHPRVALA 61 VLDGKVAVV 62 GLLGKVTSV 63 KMISAIPTL 64 GLLETTGLLAT 65TLNTLDINL 66 VIIKGLEEI 67 YLEDGFAYV 68 KIWEELSVLEV 69 LLIPFTIFM 70ISLDEVAVSL 71 KISDFGLATV 72 KLIGNIHGNEV 73 ILLSVLHQL 74 LDSEALLTL 75VLQENSSDYQSNL 76 HLLGEGAFAQV 77 SLVENIHVL 78 SLSEKSPEV 79 AMFPDTIPRV 80FLIENLLAA 81 FTAEFLEKV 82 ALYGNVQQV 83 LFQSRIAGV 84 ILAEEPIYIRV 85FLLEREQLL 86 LLLPLELSLA 87 SLAETIFIV 88 AILNVDEKNQV 89 RLFEEVLGV 90YLDEVAFML 91 KLIDEDEPLFL 92 KLFEKSTGL 93 SLLEVNEASSV 94 GVYDGREHTV 95GLYPVTLVGV 96 ALLSSVAEA 97 TLLEGISRA 98 SLIEESEEL 99 ALYVQAPTV 100KLIYKDLVSV 101 ILQDGQFLV 102 SLLDYEVSI 103 LLGDSSFFL 104 VIFEGEPMYL 105ALSYILPYL 106 FLFVDPELV 107 SEWGSPHAAVP 108 ALSELERVL 109 SLFESLEYL 110KVLEYVIKV 111 VLLNEILEQV 112 SLLNQPKAV 113 KMSELQTYV 114 ALLEQTGDMSL 115VIIKGLEEITV 116 KQFEGTVEI 117 KLQEEIPVL 118 GLAEFQENV 119 NVAEIVIHI 120ALAGIVTNV 121 NLLIDDKGTIKL 122 VLMQDSRLYL 123 KVLEHVVRV 124 LLWGNLPEI125 SLMEKNQSL 126 KLLAVIHEL 127 ALGDKFLLRV 128 FLMKNSDLYGA 129KLIDHQGLYL 130 GPGIFPPPPPQP 131 ALNESLVEC 132 GLAALAVHL 133 LLLEAVWHL134 SIIEYLPTL 135 TLHDQVHLL 136 SLLMWITQC 137 FLLDKPQDLSI 138 YLLDMPLWYL139 GLLDCPIFL 140 VLIEYNFSI 141 TLYNPERTITV 142 AVPPPPSSV 143 KLQEELNKV144 KLMDPGSLPPL 145 ALIVSLPYL 146 FLLDGSANV 147 ALDPSGNQLI 148 ILIKHLVKV149 VLLDTILQL 150 HLIAEIHTA 151 SMNGGVFAV 152 MLAEKLLQA 153 YMLDIFHEV154 ALWLPTDSATV 155 GLASRILDA 156 ALSVLRLAL 157 SYVKVLHHL 158 VYLPKIPSW159 NYEDHFPLL 160 VYIAELEKI 161 VHFEDTGKTLLF 162 VLSPFILTL 163 HLLEGSVGV

Furthermore, the TAA antigenic peptide in the context of the presentinvention is a specific ligand of MHC-class-I-molecules orMHC-class-II-molecules, preferably MHC-class-I-molecules.

The term “tumor-specific antigen” refers in the context of thisinvention to antigens that are exclusively expressed on tumor cells.They include neo-antigens that arise due to mutations, e.g. pointmutations or frame-shift mutations, in the tumor cell. Examples fortumor specific antigens are p53 or BCR-ABL.

The term “MHC” refers in the context of this invention to theabbreviation for the phrase “major histocompatibility complex”. MHC'sare a set of cell surface receptors that have an essential role inestablishing acquired immunity against altered natural or foreignproteins in vertebrates, which in turn determines histocompatibilitywithin a tissue. The main function of MHC molecules is to bind toantigens derived from altered proteins or pathogens and display them onthe cell surface for recognition by appropriate T-cells. The human MHCis also called HLA (human leukocyte antigen) complex or HLA. The MHCgene family is divided into three subgroups: class I, class II, andclass III. Complexes of peptide and MHC class I are recognized byCD8-positive T-cells bearing the appropriate TCR, whereas complexes ofpeptide and MHC class II molecules are recognized byCD4-positive-helper-T-cells bearing the appropriate TCR. Since bothtypes of response, CD8 and CD4 dependent, contribute jointly andsynergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens and corresponding TCRs isimportant in the development of cancer immunotherapies such as vaccinesand cell therapies.

The term “MHC-I” refers in the context of the present invention to MHCclass I molecules or MHC-I. The MHC I molecule consists of an alphachain, also referred to as MHC I heavy chain and a beta chain, whichconstitutes a beta 2 microglobulin molecule. The alpha chain, comprisesthree alpha domains, i.e. alpha1 domain, alpha2 domain and alpha3domain. Alpha1 and alpha2 domain mainly contribute to forming thepeptide pocket to produce a peptide ligand MHC (pMHC) complex. MHC-Itypically bind peptides that are derived from cytosolic antigenicproteins and which are degraded by the proteasome after ubiquitylationand subsequently transported through a specific transporter associatedwith antigen processing (TAP) from the cytosol to the endoplasmaticreticulum (ER). MCH I typically binds peptides of 8-12 amino acids inlength.

The term “MHC-H” refers in the context of the present invention to MHCclass II molecules or MHC-II. The MHC-II molecule consists of an alphaand a beta chain, wherein the alpha chain comprises two alpha domains,alpha1 domain, alpha2 domain and the beta chain comprises two betadomains, beta domain1 and beta domain2 MHC II typically fold in the ERin complex with a protein called invariant chain and are thentransported to late endosomal compartments where the invariant chain iscleaved by cathepsin proteases and a short fragment remains bound to thepeptide-binding groove of MHC II, termed class II-associated invariantchain peptide (CLIP). This placeholder peptide is then normallyexchanged against higher affinity peptides, which are derived fromproteolytically degraded proteins available in endocytic compartments.MHC-II typically binds peptides of 10-30 amino acids in length orpeptides of 13-25 amino acids in length.

The term “HLA” refers in the context of the present invention tomolecules which differ between different human beings in amino acidsequence. However, HLAs can be identified by an internationally agreednomenclature, the IMGT nomenclature, of HLA. The HLA-A gene is locatedon the short arm of chromosome 6 and encodes the larger, α-chain,constituent of HLA-A. Variation of HLA-A α-chain is key to HLA function.This variation promotes genetic diversity in the population. Since eachHLA has a different affinity for peptides of certain structures, greatervariety of HLAs means greater variety of antigens to be ‘presented’ onthe cell surface. Each individual can express up to two types of HLA-A,one from each of their parents. Some individuals will inherit the sameHLA-A from both parents, decreasing their individual HLA diversity.However, the majority of individuals receive two different copies ofHLA-A. The same pattern follows for all HLA groups. In other words,every single person can only express either one or two of the 2432 knownHLA-A alleles coding for currently 1740 active proteins. HLA-A*02signifies a specific HLA allele, wherein the letter A signifies to whichHLA gene the allele belongs to and the prefix “*02 prefix” indicates theA2 serotype. In MHC class I dependent immune reactions, peptides notonly have to be able to bind to certain MHC class I molecules expressedby tumor cells, they subsequently also have to be recognized by T-cellsbearing specific TCRs.

The term “target peptide” (TP) refers in the context of this inventionto a shorter peptide, part or fragment of the protein antigen ofinterest (PAI). The amino acid sequence of a target peptide comprisestypically 8-12 amino acids in length, 8-11 amino acids in length or 8-10amino acids in length. Preferably, the amino acid sequence of a targetpeptide comprises typically 8-11 amino acids in length. The targetpeptide may be bound to an MHC-I molecule or an MHC-II molecule. Whethera target peptide binds to an MHC-I or MHC-II molecule depends on thetarget peptide's natural origin, i.e. whether it is synthesized in thecytoplasm and processed in the proteasome or absorbed by endocytosis andsubsequently processed. Moreover, it depends on the length of the targetpeptide whether it will bind to the binding groove of an MHC-I or anMHC-II molecule. In one example a target peptide of a length of 8-12,8-11 or 8-10 amino acids is typically bound to a MHC-I. In anotherexample, the amino acid sequence of a target peptide may comprise 13-23amino acids in length, preferably 13-18 amino acids in length. A targetpeptide of a length of 13-25 or 13-18 amino acids is typically bound toan MHC-II.

The term “antigen complex” (AC) refers in the context of this inventionto a complex comprising an antigen that is directly or indirectly, e.g.through an MHC or peptide binding part thereof, attached to the surfaceof a carrier or a soluble multimerized MHC or peptide binding partthereof. Such a carrier can be a cell or synthetic material. If theantigen is attached to a cell, the cell may be an antigen-presentingcells (APCs), preferably a human APC. Synthetic materials for carrierscan be beads or particles, preferably microbeads, microparticles ornanoparticles. Such beads can be magnetic or paramagnetic beads. Beadsor microparticles are usually made of polymers and can be covalently ornon-covalently coated with a first member of a pair of couplingresidues. The second member of the pair of coupling residues iscovalently or non-covalently coupled to the MHC or peptide binding partthereof. A preferred pair of first and second coupling residuescomprises streptavidin and biotin member. The skilled person is aware ofother pairs of coupling residues. Accordingly, in a preferred embodimentthe carrier may be coated with streptavidin which will allow theimmobilization of MHC molecules or peptide binding parts thereof thatcomprise a biotin moiety. Conversely, a carrier coated with biotinallows the immobilization of MHC molecules or peptide binding partsthereof that comprise a streptavidin moiety. A soluble multimerized MHCor peptide binding part thereof may comprise two or more MHCs, whereineach is covalently or non-covalently, preferably covalently coupled to athird member of a pair of coupling residues and a fourth member of apair or coupling residues, wherein the fourth member has at least twobinding sites for the third member, preferably 3, 4, 5, 6, 7, or 8binding sites and particularly preferred 4 binding sites. Biotin is apreferred third member of a pair of coupling residues and streptavidinis a preferred fourth member of a pair of coupling residues.Streptavidin has four binding sites for biotin. Thus, if MHC peptidecomplexes comprising biotin are contacted with streptavidin a solubletetramer will form in which four peptide loaded MHCs (or peptide bindingfragments thereof) are non-covalently bound to streptavidin. Thus, in apreferred embodiment the soluble multimerized MHC or peptide bindingfragment thereof is a complex comprising four MHC peptide complexes,wherein each of the MHC peptide complex is attached covalently to onebiotin, which are in turn bound non-covalently to streptavidin.

The term “pair of coupling residues” refers to two entities thatspecifically and non-covalently bind to each other with high affinity.Preferably, the K_(d) is less than 10⁻¹⁰ mol/L, more preferably lessthan 10⁻¹¹ mol/L, more preferably less than 10-12 mol/L and even morepreferably less than 10⁻¹³ mol/L. Preferably, at least one of themembers of a binding pair has a molecular weight below 500 g/mol/. Sucha molecule can be attached covalently to one chain of the MHC or peptidebinding fragment thereof without interfering with the ability of the MHCto interact with a TCR. Preferred pairs of coupling residues arebiotin-streptavidin, and biotin-avidin. Alternatively, one member of apair of coupling residues can be a protein that is fused to one chain ofan MHC. Examples include chitin binding protein (CBP), maltose bindingprotein (MBP), Strep-tag glutathione-S-transferase (GST), poly(His) tag,V5-tag, Myc-tag, HA-tag, Spot-tag, T7-tag and NE-tag. The other memberof the pair is determined by the respective protein tag, i.e. chitin,maltose, biotin, glutathione, metal matrix, e.g. Ni-matrix, or anantibody that specifically binds to the V5-, Myc-, HA-, Spot-, T7- orNE-tag.

The term “similar protein antigen” (SPA) refers in the context of thisinvention to a protein or a portion of a protein or a protein complexthat comprises an epitope bound by the paratope of an antigen bindingprotein. The amino acid sequence of the SPA is determined by the PAI.The amino acid sequence of the SPA differs in at least one amino acidfrom the amino acid sequence of the given PAI, i.e. the PAI of interest.It serves the purpose of identifying antigen binding proteins that bindthe PAI and at the same time the SPA, i.e. that do not exhibit thedesired specificity and/or selectivity to the PAI. Such antigen bindingproteins may elicit off-tumor/off target toxicity. For a given PAI andantigen binding protein combination, the SPA falls into one of threecategories:

(1) Similar Amino Acid Sequence, Identical Epitope:

If the amino acids of the SPA that differ with respect to the PAI do notcontribute to the epitope bound by a given PAI-specific antigen bindingprotein then the antigen binding protein will bind with the sameaffinity both to the PAI and the SPA. An antigen binding protein withthis property will be counter selected by the methods of the presentinvention.

(2) Similar Amino Acid Sequence, Similar Epitope:

If at least one of the amino acids of the SPA that differ with respectto the PAI contributes to the epitope bound by a given PAI-specificantigen binding protein than the antigen binding protein will bind witha different affinity to the PAI and the SPA. An antigen binding proteinthat exhibits significantly lower binding to the SPA than to the PAI maybe selected by the methods of the present invention. In this respectsignificantly lower binding means that the difference between thebinding to the PAI and the SPA is at least 2-fold, at least 3-fold, atleast 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, atleast 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, atleast 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, atleast 70-fold, at least 100-fold, at least 200-fold, preferably at least50-fold, more preferably at least 100-fold at identical concentration ofthe PAI and the SPA.

(3) Similar Amino Acid Sequence, Different Epitope:

If the diverging amino acids are located at positions that contributesto the epitope bound by a given PAI-specific antigen binding proteinthen the antigen binding protein may not bind to the SPA at all. Anantigen binding protein with this property will be selected by themethods of the present invention.

The amino acid sequences of the SPAs are generally based on the aminoacid sequences of naturally occurring proteins, since such proteins maybe expressed on healthy tissue of a tumor patient. Preferably, the SPAis a naturally occurring protein or a fragment thereof. In particularthe SPA is present in the same species as the PAI. Thus, it is desiredthat the SPAs included in the method of the invention have amino acidsequences that allow identification and counterselection of antigenbinding proteins in category (1) and (2). The SPA is only likely toallow the counterselection of unsuitable antigen binding proteins if itsamino acid sequence is closely related to the amino acid sequence of thePAI. It is, thus, preferred that the amino acid sequence of the SPA usedin the method of the present invention has a similarity to the aminoacid sequence of the PAI of at least 50%, at least 60%, at least 70%, atleast 80%, of at least 90% or at least 95%. Thus, in a preferredembodiment the SPA differs by 1-20, more preferably by 2-10 amino acidsfrom the amino acid sequence of the PAI. It is preferred that the SPA,in particular the target similar peptide (TSP) used in the method of theinvention is expressed on healthy tissue, preferably with more than 10copies per cell, preferably more than 20 copies per cell, preferablymore than 50 copies per cell and preferably more than 100 copies percell. The relative strength of expression can be determined by a varietyof art known methods including FACS analysis of healthy and diseasedcells with fluorescently labeled antigen binding proteins or massspectrometry. Gene expression analysis can also be performed using RNAsequencing approaches. Another criteria for the selection of a SPA to beused in the method of the invention is its frequency of presentation onprimary normal tissues. The frequency describes how often a SPA ispresented on normal, i.e. a healthy tissue—in contrast to the copynumber which defines the number of SPAs of a given healthy tissue, forexample a cell. Together with the copy number the frequency is animportant criterion to select a SPA for a given PAI. The higher thesimilarity to the PAI and the higher the presentation frequency and thecopy number per cell (CpC) on normal tissues, the higher the relevanceof a SPA.

The term “target similar peptide” (TSP) refers in the context of thisinvention to a shorter peptide, part or fragment of the SPA. The aminoacid sequence of a TSP comprises typically 8 to 16 amino acids inlength. The TSP is typically MHC presented. Similarly to the SPA, theTSP has a similarity to the amino acid sequence of the TP of at least50%, at least 60%, at least 70%, at least 80%, at least 90%. The TSPstypically have a length of 8 to 16 amino acids, such as 8-12, preferablyof 8 to 11 amino acids. In particular, TSPs have a length of 8 to 11 or8-12 amino acids when they bind to MHC-I. In another example, TSPstypically have a length of 13 to 25 amino acids when they bind toMHC-II. TSPs may differ in one or more amino acids from the TP in aslong as they meet the similarity scores outlined above and which may bedetermined as explained below. Thus, if the amino acid sequence of a TSPof 8 amino acids length is aligned to a given TP it may comprise between1 to 8 amino acids that are similar to the corresponding amino acids ofthe TP. The other amino acids may be identical or dissimilar to the TP.Accordingly, a TSP of 9 amino acids length may comprise between 1 to 9amino acids that are similar to the corresponding amino acids of the TP;a TSP of 10 amino acids length may comprise between 1 to 10 amino acidsthat are similar to the corresponding amino acids of the TP; a TSP of 11amino acids length may comprise between 1 to 11 amino acids that aresimilar to the corresponding amino acids of the TP, a TSP of 12 aminoacids length may comprise between 1 to 12 amino acids that are similarto the corresponding amino acids of the TP. If the TSP is bound to MHCII it typically has a length of 13 to 25 amino acids and accordingly, aTSP of 13 amino acids length may comprise between 1 to 13 amino acidsthat are similar to the corresponding amino acids of the TP; a TSP of 14amino acids length may comprise between 1 to 14 amino acids that aresimilar to the corresponding amino acids of the TP; a TSP of 15 aminoacids length may comprise between 1 to 15 amino acids that are similarto the corresponding amino acids of the TP, a TSP of 16 amino acidslength may comprise between 1 to 16 amino acids that are similar to thecorresponding amino acids of the TP, a TSP of 17 amino acids length maycomprise between 1 to 17 amino acids that are similar to thecorresponding amino acids of the TP; a TSP of 18 amino acids length maycomprise between 1 to 18 amino acids that are similar to thecorresponding amino acids of the TP; a TSP of 19 amino acids length maycomprise between 1 to 19 amino acids that are similar to thecorresponding amino acids of the TP, a TSP of 20 amino acids length maycomprise between 1 to 20 amino acids that are similar to thecorresponding amino acids of the TP. Another criteria for the selectionof a TSP to be used in the method of the present invention is itsfrequency of presentation on primary normal tissues. The frequency ofpresentation describes how often a TSP is presented on normal, i.e. ahealthy tissue—in contrast to the copy number which defines the numberof TSPs on different samples of a given healthy tissue, e.g. if acertain TSP is detected on 6 out of 12 samples of adipose tissue is hasa frequency of presentation of 50%. The frequency of presentation of agiven TSP can be determined by art known methods including MS analysisas used in Example 4 (see FIGS. 6 to 8, which indicate frequency ofpresentation for three different TSPs). Thus, it is preferred that a TSPis used in the method of the invention, which has a frequency ofpresentation of at least 10% in at least one healthy tissue, preferably,at least 20% in at least one healthy tissue, more preferably at least30% in at least one healthy tissue. Preferably, the selected TSP ispresented in at least one, preferably at least two, more preferably atleast three healthy tissues. These tissues are preferably selected fromthose that were analyzed regarding their presentation of TSP1, and TSP2,respectively, in FIGS. 6 and 7.

Together with the copy number the frequency is an important criterion toselect a TSP for a given TP. The higher the similarity to the TP and thehigher the presentation frequency and CpC on normal tissues, the higherthe relevance of a TSP.

The term “irrelevant antigen complex” (IAC) refers in the context ofthis invention to an AC comprising an irrelevant protein antigen (IPA).Such an AC can be, e.g. an APC, or a multimerized MHC-peptide complexthat is soluble or MHC-peptide complexes immobilized on a carrier. Theirrelevant protein antigen is defined in the following.

The term “irrelevant protein antigen” (IPA) refers in the context ofthis invention to a protein antigen which is not bound by a selectedTCR. TCRs are screened for binding their respective target peptides.Upon binding of a TCR to its target peptide a desired immune reaction orT-cell mediated immune response is triggered. Such a desired immuneresponse will not be triggered by an irrelevant peptide because anirrelevant peptide will not be bound by a TCR in the screening. The IPAmay be a protein encoded by a housekeeping gene. Typically, the IPA hasa similarity to the amino acid sequence of the TP of at least less than50%, at least less than 40%, at least less than 30%, at least less than20%, at least less than 10%.

The term “irrelevant peptide” (IP) refers in the context of thisinvention to a shorter peptide, part or fragment of the IPA. The aminoacid sequence of an IP comprises typically 8-16 amino acids in length.Such an IP is typically bound to MHC-I. In some examples, when an IP isbound to a MHC-II, an IP may comprise 13-25 amino acids in length. An IPmay also comprise 13-18 amino acids in length when bound to a MHC-II.The IP may be encoded by a housekeeping gene.

“Housekeeping genes” in the context of this invention are typicallyconstitutive genes that are required for the maintenance of basiccellular function and are expressed in all cells of an organism undernormal and pathophysiological conditions. Although some housekeepinggenes are expressed at relatively constant rates in mostnon-pathological situations, the expression of other housekeeping genesmay vary depending on experimental conditions. Housekeeping genesaccount for majority of the active genes in the genome, and theirexpression is obviously vital to survival. The housekeeping geneexpression levels are fine-tuned to meet the metabolic requirements invarious tissues. Examples for housekeeping genes are listed(non-exhaustive) as follows: Transcription factor, translation factors,repressor molecules, RNA splicing molecules, RNA binding proteins,ribosomal proteins, mitochondrial ribosomal proteins, RNA polymerases,protein processing genes, heat shock proteins, histone, cell cycle,apoptosis, oncogenes, DNA repair, DNA replication, metabolism involvedgenes, e.g. genes involved in carbohydrate metabolism, citrate cycle,lipid metabolism, amino acid metabolism, NADH dehydrogenase, cytochromeC oxidase, ATPase, lysosome, proteasome, ribonuclease, thioreductases,receptors, channels, transporters, HLA/immunoglobulin/cell recognition,kinases, cytoskeletal, growth factors, tumor necrosis factor α.Similarly to the IPA, the IP has a similarity to the amino acid sequenceof the TP of at least less than 50%, at least less than 40%, at leastless than 30%, at least less than 20%, at least less than 10%,preferably at least less than 30%, at least less than 20%, at least lessthan 10%.

The term “amino acid sequence identity” refers in the context of thisinvention to the percentage of sequence identity and is determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the sequence in the comparison window cancomprise additions or deletions (i.e. gaps) as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The term “identical” refers in the context of two or more polypeptide ornucleic acid sequences, refers to two or more sequences or subsequencesthat are the same, i.e. comprise the same sequence of amino acids ornucleic acids. Sequences are “substantially identical” to each other ifthey have a specified percentage of amino acid residues that are thesame (e.g., at least 70%, at least 75%, at least 80, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity over a specifiedregion), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. These definitions also refer to the complement of atest sequence. Accordingly, the term “at least 80% sequence identity” isused throughout the specification with regard to polypeptide andpolynucleotide sequence comparisons. This expression preferably refersto a sequence identity of at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% to the respective reference polypeptide or tothe respective reference polynucleotide.

The term “sequence comparison” refers in the context of this inventionto the process wherein one sequence acts as a reference sequence, towhich test sequences are compared. When using a sequence comparisonalgorithm, test and reference sequences are inputted into a computer, ifnecessary subsequence coordinates are designated, and sequence algorithmprogram parameters are designated. Default program parameters arecommonly used, or alternative parameters can be designated. The sequencecomparison algorithm then calculates the percent sequence identities orsimilarities for the test sequences relative to the reference sequence,based on the program parameters. In case where two sequences arecompared and the reference sequence is not specified in comparison towhich the sequence identity percentage is to be calculated, the sequenceidentity is to be calculated with reference to the longer of the twosequences to be compared, if not specifically indicated otherwise. Ifthe reference sequence is indicated, the sequence identity is determinedon the basis of the full length of the reference sequence indicated bySEQ ID, if not specifically indicated otherwise.

In a sequence alignment, the term “comparison window” refers to thosestretches of contiguous positions of a sequence which are compared to areference stretch of contiguous positions of a sequence having the samenumber of positions. It is preferred that the entire length of the PAI,preferably the TP is used as comparison window in the alignment with theSPA and TSP, respectively. If the TP is, e.g. a 10 amino acid long MHC 1presented peptide the similarity is determined in a comparison window of10 amino acids. In this case a 9 amino acids long SPA with one aminoacid mismatch has an identity of 80% to the given TP.

Methods of alignment of sequences for comparison are well known in theart. Optimal alignment of sequences for comparison can be conducted, forexample, by the local homology algorithm of Smith and Waterman (Adv.Appl. Math. 2:482, 1970), by the homology alignment algorithm ofNeedleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search forsimilarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA85:2444, 1988), by computerized implementations of these algorithms(e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Ausubelet al., Current Protocols in Molecular Biology (1995 supplement)).Algorithms suitable for determining percent sequence identity andsequence similarity are the BLAST and BLAST 2.0 algorithms, which aredescribed in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977), andAltschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http://www.ncbi.nlm nih.gov/).This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The term “similarity” of two amino acid sequences takes intoconsideration the relatedness of two amino acids at a given position(see, for example below Table 4). The similarity of two amino acidsequences, e.g. in a TP and a TSP, can be determined using the BLASTalgorithm, which performs a statistical analysis of the similaritybetween two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad.Sci. USA 90:5873-87, 1993). Preferred settings for such an alignmentare: wordlength of 3, and expectation (E) of 10, and the use of theBLOSUM62 or PMBEC scoring matrix (Kim et al. 2009 BMC Bioinformatics),preferably the PMBEC scoring matrix is used in the determination of thesimilarity. These matrices quantify amino acid similarity based forexample on evolutionary or functional similarity between amino acids,which correlate well with the similarity according to physicochemicalparameters. For each substitution of an amino acid in a given TPsequence a score (decimal value) can be calculated by using thesematrices, which indicates the similarity of the amino acid in the TPsequence with the substituted amino acid in the TSP sequence. Multiplesubstitutions can be considered by summing up the effect (scores) ofindividual substitutions in the TP sequence. By definition, the maximumscore which can be achieved for a TSP is provided by the unsubstitutedTP sequence, whereas any substitution leading to a TSP will be penalizedin the scoring matrix and ultimately lead to a lower score of a TSP.This maximum score is however dependent on the length and amino acidsequence of the TP (i.e. different TP sequences will have differentmaximum scores). Typically, a longer amino acid sequences results in ahigher score. However, the score of a TP depends on the score allottedto the amino acids it consists of. In order to be able to calculate andcompare the similarity of a TSP in reference to a TP without consideringthe difference of maximum scores of distinct TP sequences the respectivedecimal values are converted, which are the result of calculating thesimilarity of a TSP in reference to a TP, into a percentage scorewherein the maximum score of a TP sequence will therefore, always be100%.

Another measure of similarity provided by the BLAST algorithm is thesmallest sum probability (P(N)), which provides an indication of theprobability by which a match between amino acid sequences would occur bychance. For example, an amino acid is considered similar to a referencesequence if the smallest sum probability in a comparison of the testamino acid to the reference amino acid is less than about 0.2, typicallyless than about 0.01, and more typically less than about 0.001.Semi-conservative and especially conservative amino acid substitutions,wherein an amino acid is substituted with a chemically related aminoacid are preferred. Typical substitutions are among the aliphatic aminoacids, among the amino acids having aliphatic hydroxyl side chain, amongthe amino acids having acidic residues, among the amide derivatives,among the amino acids with basic residues, or the amino acids havingaromatic residues. Typical semi-conservative and conservativesubstitutions are indicated in below Table 4.

TABLE 4 Amino acids and conservative and semi- conservativesubstitutions, respectively. Semi-conservative Amino acid Conservativesubstitution substitution A G; S; TN; V; C C A; V; L M; I; F; G D E; N;Q A; S; T; K; R; H E D; Q; N A; S; T; K; R; F W; Y; L; M; H I; V; A G AS; N; T; D; E; N; Q H Y; F; K; R L; M; A I V; L; M; A F; Y; W; G K R; HD; E; N; Q; S; T; A L M; I; V; A F; Y; W; H; C M L; I; V; A F; Y; W; C;N Q D; E; S; T; A; G; K; R P V; I L; A; M; W; Y; S; T; C; F Q N D; E; A;S; T; L; M; K; R R K; H N; Q; S; T; D; E; A S A; T; G; N D; E; R; K T A;S; G; N; V D; E; R; K; I V A; L; I M; T; C; N W F; Y; H L; M; I; V; C YF; W; H L; M; I; V; C

Changing from A, F, H, I, L, M, P, V, W or Y to C is semi-conservativeif the new cysteine remains as a free thiol. Furthermore, the skilledperson will appreciate that glycines at sterically demanding positionsshould not be substituted and that P should not be introduced into partsof the protein which have an alpha-helical or a beta-sheet structure.

The term “detectable label” refers in the context of this invention to amolecule which labels a different molecule or a cell by allowing thisdifferent molecule to be selected due to a property or specificcharacteristics the label exerts. Molecules that are eligible forlabelling are proteins, DNA or RNA or synthetic materials such as beadsor other suitable materials. Regarding proteins, labeling strategiesresult in the covalent attachment of different molecules, includingbiotin, reporter enzymes, fluorophores, magnetic labels and radioactiveisotopes, to the target protein or peptide or nucleotide sequence.Single-cell can be labeled using short DNA or RNA barcode ‘tags’ toidentify reads that originate from the same cell in a sequencingexperiment. Labels may be a fluorescent label, e.g. xanthens, acridines,oxazines, cynines, styryl dyes, coumarines, porphines,metal-ligand-complexes, fluorescent proteins, nanocrystals, perylenesand phtalocyanines, phycoerythrin (SA-PE), streptavidin-allophycocyanin(SA-APC) or streptavidin-brilliant-violet 421 (SA-BV421); RNA-barcodesor DNA-barcodes or a radioactive label. A radioactive label is typicallya molecule wherein one or more atoms are replaced by the radioactivecounterparts, i.e. radio isotopes. Proteins, peptides, DNA or RNA may belabeled radioactively. Magnetic labels may comprise magnetic beads ormagnetic nanoparticles whjch can be coated with e.g. antibodies againsta particular surface antigen. Magnetic labels may be used inmagnetic-activated cell sorting (MACS).

The term “detectably different” refers in the context of this inventionto a scenario wherein two labels are present but may only be differentin the signal they are emitting. For example, two cells may be labelledwith a fluorescent label and are thus, not distinguishable by thecharacteristics of the label as such, i.e. the fluorescence. However,the fluorescence label attached to one cell may signal in red whereinthe fluorescence signal attached to the second cell may signal in green.The two labels of the exemplified cells are thus, detectably different.

The term “flow cytometry analysis” refers in the context of thisinvention to a sorting technique comprising the measurement of chemicaland physical properties of a specific cell populations or cellsubpopulations in a sample. The sample usually is a suspension and isadjusted to result in a flow of one cell at a time through a detectionunit, typically a laser beam that excites a fluorophore and a lightdetector. The detected signal, e.g. light scattered by the flow throughof the cell, is characteristic to the cell, i.e. its components.Multiple cells can be analyzed by this technique in a short period oftime. Routine applications of flow cytometry are cell counting, cellsorting, determination of cell characteristic and functions, diagnosisof diseases, e.g. cancer, detection of biomarkers or detection ofmicroorganisms. A popular flow cytometry technique is fluorescenceactivated cell sorting (FACS). The FACS technique harnesses the abilityto label a target cell/cells with fluorescent dye tags or labels whichallows for the cell sorting based on the individual labeling profile ofa particular cell population.

The term “magnetic-activated cell sorting” (MACS) refers to a sortingtechnique that harnesses functional micro- or nanoparticles that areconjugated with antibodies corresponding to particular cell surfaceantigens. Under application of a magnetic field gradient, themagnetically targeted cells can be separated in either a positive ornegative fashion with the respect to the antigen employed. A skilledperson is well aware of the different kind of sorting analyses.

The term “specifically binding” refers in the context of this inventionto the binding of an antigen binding protein or fragments thereof, e.g.an antibody or fragments thereof or a TCR or fragments thereof, to aspecific binding site of its target when the target comprises specificand non-specific binding sites. However, sometimes binding of a proteinto closely related proteins is unavoidable, then the actual binding tothe target may be specific but the protein is deemed to be non-specificin relation to the intended target binding. An antigen binding proteinor a fragment thereof of the present invention is considered to bindspecifically to a given antigen, if it binds to the antigen with a K_(d)of 10⁻⁵ M or less when measured by surface plasmon resonance (SPR) atRT. The dissociation constant (K_(d)) for the target to which thebinding moiety specifically binds is at least 2-fold, at least 5-fold,at least 7-fold, 10-fold, preferably at least 20-fold, more preferablyat least 50-fold, even more preferably at least 100-fold, 200-fold,500-fold or 1000-fold lower than the dissociation constant (K_(d)) forthe target to which the binding moiety of the antigen binding protein orfragment thereof does not bind specifically, for example the similarprotein antigen (SPA), preferably the target similar peptide (TSP).

Typically, if the antigen binding protein of the present invention thatspecifically binds to a given TP is a TCR or a fragment thereof it has aK_(d) in the range of 3×10⁻⁵ to 1×10⁻⁷, 2×10⁻⁵ to 5×10⁻⁷, 1×10⁻⁵ to1×10⁻⁶⁰r 5×10⁻⁶ to 1×10⁻⁶. In this situation, it is preferred that theantigen binding protein at the same time has a K_(d) for the TP, that isat least 2-fold lower, at least 5-fold, at least 7-fold, 10-fold,preferably at least 20-fold, more preferably at least 50-fold, even morepreferably at least 100-fold, 200-fold, 500-fold or 1000-fold lower thanthe K_(d) for the target to which the binding moiety of the antigenbinding protein does not bind specifically, for example the TSP. Thus,for example a selected TCR may bind to the TP with a K_(D) of 1×10⁻⁶ andto the TSP with a K_(D) of 1×10⁻⁵.

Typically, if the antigen binding protein of the present invention thatspecifically binds to a given TP is an affinity maturated TCR or afragment thereof or a soluble molecule in a bispecific format orfragment thereof, such as a TCER® molecule or a fragment thereof, theK_(D) in the range of 9×10⁻⁹ to 1×10⁻¹², 8×10⁻⁹ to 5×10⁻¹², 7×10⁻⁹ to1×10⁻¹¹, 6×10⁻⁹ to 2×10⁻¹¹, 5×10⁻⁹ to 5×10⁻¹¹, 4×10⁻⁹ to 8×10⁻¹¹, 3×10⁻⁹to 1×10⁻¹⁰. In this situation, it is preferred that the antigen bindingprotein at the same time has a K_(d) for the TP, that is at least 2-foldlower, at least 5-fold, at least 7-fold, 10-fold, preferably at least20-fold, more preferably at least 50-fold, even more preferably at least100-fold, 200-fold, 500-fold or 1000-fold lower than the K_(d) for thetarget to which the binding moiety of the antigen binding protein doesnot bind specifically, for example the TSP. Molecules in the bispecificformat referred to herein as “TCER®” molecules or “TCER®” typicallycomprise a first polypeptide that specifically binds to a surfacemolecule on a T cell and a second polypeptide that specifically binds toa MHC-peptide complex.

Typically, if the antigen binding protein of the present invention is anantibody or a fragment thereof or a B-cell that specifically binds to agiven PAI, the K_(D) is in the range of 9×10⁻⁹ to 1×10⁻¹², 8×10⁻⁹ to5×10⁻¹², 7×10⁻⁹ to 1×10⁻¹¹, 6×10⁻⁹ to 2×10⁻¹¹, 5×10⁻⁹ to 5×10⁻¹¹, 4×10⁻⁹to 8×10⁻¹¹, 3×10⁻⁹ to 1×10⁻¹°. In this situation, it is preferred thatthe antigen binding protein at the same time has a K_(d) for the PAI,that is at least 2-fold lower, at least 5-fold, at least 7-fold,10-fold, preferably at least 20-fold, more preferably at least 50-fold,even more preferably at least 100-fold, 200-fold, 500-fold or 1000-foldlower than the K_(d) for the target to which the binding moiety of theantigen binding protein does not bind specifically, for example the SPA.

In some instances, in particular in context of TCRs, if the antigenbinding protein of the present invention, in particular in context of aTCR, specifically binds to a given TP might be determined by using afunctional assay, for example, in a TCR activation assay, such as anIFNγ-release assay. Accordingly, a specific binding may be characterizedby a response, such as signal that is detected for a TP is more than30%, more than 40%, more than 50%, more than 60%, more than 70%, morethan 80%, more than 90%, more than 100% of the response, i.e. thesignal, obtained for the TSP in such an assay.

The term “selectively binding” refers in the context of this inventionto the characteristic of an antigen binding protein, such as a TCR orantibody, to selectively recognize or bind to preferably only onespecific epitope and preferably shows no or substantially no binding (nocross-reactivity) to another epitope, peptide or protein. Assessing thethreshold of epitope binding by flow cytometry can be assessed by usingnon-tetramer stained controls. The gates can be set according to thenon-tetramer stained control in a way that <0.01% cells appear in thisgate. This gate can be applied to a sample from the same donor that hasbeen stained with tetramer of interest. Cells which appear in this gateare considered to bind selectively to the epitope of interest.

The term “T-cell receptor” (TCR) refers in the context of this inventionto a heterodimeric cell surface protein of the immunoglobulinsuper-family, which is associated with invariant proteins of the CD3complex involved in mediating signal transduction. TCRs exist in αβ andγδ forms, which are structurally similar but have quite distinctanatomical locations and probably functions. The extracellular portionof native heterodimeric αβ TCR and γβ TCR each contain two polypeptides,each of which has a membrane-proximal constant domain, and amembrane-distal variable domain. Each of the constant and variabledomains include an intra-chain disulfide bond. The variable domainscontain the highly polymorphic loops analogous to the complementaritydetermining regions (CDRs) of antibodies. The use of TCR gene therapyovercomes a number of current hurdles. It allows equipping the subjects'(patients') own T-cells with desired specificities and generation ofsufficient numbers of T-cells in a short period of time, avoiding theirexhaustion. The TCR will be transduced into potent T-cells (e.g. centralmemory T-cells or T-cells with stem cell characteristics), which mayensure better persistence, preservation and function upon transfer.TCR-engineered T-cells will be infused into cancer patients renderedlymphopenic by chemotherapy or irradiation, allowing efficientengraftment but inhibiting immune suppression. Native alpha-betaheterodimeric TCRs have an alpha chain and a beta chain. Each alphachain comprises variable, joining and constant regions, and the betachain also usually contains a short diversity region between thevariable and joining regions, but this diversity region is oftenconsidered as part of the joining region. The constant, or C, regions ofTCR alpha and beta chains are referred to as TRAC and TRBC respectively(Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10). Eachvariable region, herein referred to as alpha variable domain and betavariable domain, comprises three “complementarity determining regions”(CDRs) embedded in a framework sequence, one being the hypervariableregion named CDR3. The alpha variable domain CDRs are referred to asCDRa1, CDRa2, CDRa3, and the beta variable domain CDRs are referred toas CDRb1, CDRb2, CDRb3. There are several types of alpha chain variable(Valpha) regions and several types of beta chain variable (Vbeta)regions distinguished by their framework, CDR1 and CDR2 sequences, andby a partly defined CDR3 sequence. The Valpha types are referred to inIMGT nomenclature by a unique TRAY number, Vbeta types are referred inIMGT nomenclature to by a unique TRBV number (Folch and Lefranc, (2000),Exp Clin Immunogenet 17(1): 42-54; Scaviner and Lefranc, (2000), ExpClin Immunogenet 17(2): 83-96; LeFranc and LeFranc, (2001), “T-cellReceptor Factsbook”, Academic Press). For more information onimmunoglobulin antibody and TCR genes see the internationalImMunoGeneTics information System®, Lefranc M-P et al (Nucleic AcidsRes. 2015 January; 43 (Database issue): D413-22; andhttp://www.imgt.org/). A conventional TCR antigen-binding site usuallyincludes six CDRs, comprising the CDR set from each of an alpha and abeta chain variable region, wherein CDR1 and CDR3 sequences are relevantto the recognition and binding of the peptide antigen that is bound tothe MHC protein and the CDR2 sequences are relevant to the recognitionand binding of the MHC protein. Analogous to antibodies, TCRs compriseframework regions which are amino acid sequences interposed betweenCDRs, i.e. to those portions of TCR alpha and beta chain variableregions that are relatively conserved among different TCRs. The alphaand beta chains of a TCR each have four FRs, herein designated FR1-a,FR2-a, FR3-a, FR4-a, and FR1-b, FK3 b, FR4-b, respectively. Accordingly,the alpha chain variable domain may thus be designated as(FR1-a)-(CDRa1)-(FR2-1)-(CDRa2)-(1-R3-a)-(CDRa3)-(FR4-a) and the betachain variable domain may thus be designated as(FR1-b)-(CDRb1)-(FR2-b)-(CDRb2)-(FR3-b)-(CDRb3)-(FR4-b).

A “disease caused by a virus or bacteria” may also be referred to as aviral or bacterial infection. In the context of the present invention,the virus causing the disease may be selected from the group constitutedof for example, human immunodeficiency viruses (HIV), HumaneCytomegalievirus (HCMV), cytomegalovirus (CMV), human papillomavirus(HPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), humanpapillomavirus infection (HPV), Epstein-Barr virus (EBV), Influenzavirus, preferably human immunodeficiency viruses (HIV). In the contextof the present invention, the bacteria causing the disease may beMycobacterium tuberculosis. The disease caused by this bacterium is,thus, tuberculosis. It will be understood by the skilled in the art,that when the antigen binding protein targets a viral antigenic peptide,for instance, a HIV peptide, the antigen binding protein may be for usein the treatment of HIV. Accordingly, an antigen binding proteintargeting the viral or bacterial antigenic peptide TA-C is thus,suitable for use in the treatment of virus or bacteria from which saidantigenic viral or bacterial antigenic peptide, is derived.

The term “immune disease” refers in the context of this invention to adisease triggered by the immune system. The term “disease” refers to anabnormal condition, especially an abnormal medical condition such as anillness or injury, wherein a tissue, an organ or an individual is notable to efficiently fulfil its function anymore. In contrast, healthytissue, organs or individuals are referred to herein if no abnormalconditions are present and the tissue, organ or individual is withoutpathological finding. In healthy tissues random migration of cells isabsent, cells adhere to each other in structures characterizing thetissue and assist in its function. No metastasis is present in healthytissue. Typically, but not necessarily, a disease is associated withspecific symptoms or signs indicating the presence of such disease. Thepresence of such symptoms or signs may thus, be indicative for a tissue,an organ or an individual suffering from a disease. An alteration ofthese symptoms or signs may be indicative for the progression of such adisease. A progression of a disease is typically characterized by anincrease or decrease of such symptoms or signs which may indicate a“worsening” or “bettering” of the disease. The “worsening” of a diseaseis characterized by a decreasing ability of a tissue, organ or organismto fulfil its function efficiently, whereas the “bettering” of a diseaseis typically characterized by an increase in the ability of a tissue, anorgan or an individual to fulfil its function efficiently. A tissue, anorgan or an individual being at “risk of developing” a disease is in ahealthy state but shows potential of a disease emerging. Typically, therisk of developing a disease is associated with early or weak signs orsymptoms of such disease. In such case, the onset of the disease maystill be prevented by treatment. Examples of a disease include but arenot limited to infectious diseases, traumatic diseases, inflammatorydiseases, cutaneous conditions, endocrine diseases, intestinal diseases,neurological disorders, joint diseases, genetic disorders, autoimmunediseases, and various types of cancer. Healthy tissue as defined hereinusually comprises or consists of healthy cells.

The term “neoplastic disease” refers in the context of this invention todiseases characterized by an abnormal growth of cells, also known as atumor. Neoplastic diseases are conditions that cause tumor growth.Malignant tumors are cancerous and can grow slowly or quickly and carrythe risk of metastasis or spreading to multiple tissues and organs. By“tumor” is meant an abnormal group of cells or tissue that grows by arapid, uncontrolled cellular proliferation and continues to grow afterthe stimuli that initiated the new growth cease. Tumors show partial orcomplete lack of structural organization and functional coordinationwith the normal tissue, and usually form a distinct mass of tissue,which may be either benign or malignant. A neoplastic disease may resultin cancer, wherein exemplified cancer type diseases include but are notlimited to Basal cell carcinoma, Bladder cancer, Bone cancer, Braintumor, Breast cancer, Burkitt lymphoma, Cervical cancer, Colon Cancer,Cutaneous T-cell lymphoma, Esophageal cancer, Retinoblastoma, Gastric(Stomach) cancer, Gastrointestinal stromal tumor, Glioma, Hodgkinlymphoma, Kaposi sarcoma, Leukemias, Lymphomas, Melanoma, Oropharyngealcancer, Ovarian cancer, Pancreatic cancer, Pleuropulmonary blastoma,Prostate cancer, Throat cancer, Thyroid cancer, and Urethral cancer.

The term “treating” or “treatment” refers in the context of the presentinvention to a therapeutic use, e.g. for a subject in need thereof, i.e.suffering a disease or disorder) and means reversing, alleviating,inhibiting the progress of one or more symptoms of such a disease,disorder or condition. Therefore, treatment does not only refer to atreatment that leads to a complete cure of the disease, but also totreatments that slow down the progression of the disease and/or prolongthe survival of the subject.

The term “immune cell specific surface marker” refers in the context ofthis invention to cell surface antigens, which serve as monograms tohelp identify and classify immune cells. Examples of such markers thatcharacterize different T-cell subtypes are indicated in Table 1 above.The majority of immune cell specific surface markers are molecules orantigens within cell's plasma membrane. These molecules serve not onlyas markers but they also have key functional roles.

The term “growth factor” or “differentiation factor” is used in thecontext of this invention interchangeably and refers to molecules thatare capable of stimulation cellular growth, cell proliferation andcellular differentiation and regulate multiple cellular processes.Growth factors are usually proteins or steroid hormones. Examples ofprevailing molecules are listed in the following (non-exhaustiveenumeration): Growth factors, such as colony stimulating factor (CSF),Macrophage colony-stimulating factor (M-CSF), Granulocytecolony-stimulating factor (G-CSF) and Granulocyte macrophagecolony-stimulating factor (GM-CSF); epidermal growth factor (EGF);erythropoietin (EPO); fibroblast growth factor (FGF); foetal bovinesomatotropin (FBS); hepatocyte growth factor (HGF); insulin; insulinlike growth factor (IGF); interleukins; neuregulins; neutrotrophins;T-cell growth factor (TCGF); transforming growth factor (TGF); tumornecrosis factor alpha (TNFα); vascular endothelial growth factor (VEGF).

The term “nucleic acid” refers in the context of this invention tosingle or double-stranded oligo- or polymers of deoxyribonucleotide orribonucleotide bases or both. Nucleotide monomers are composed of anucleobase, a five-carbon sugar (such as but not limited to ribose or2′-deoxyribose), and one to three phosphate groups. Typically, a nucleicacid is formed through phosphodiester bonds between the individualnucleotide monomers. In the context of the present invention, the termnucleic acid includes but is not limited to ribonucleic acid (RNA) anddeoxyribonucleic acid (DNA) molecules but also includes synthetic formsof nucleic acids comprising other linkages (e.g., peptide nucleic acidsas described in Nielsen et al. (Science 254:1497-1500, 1991). Typically,nucleic acids are single- or double-stranded molecules and are composedof naturally occurring nucleotides. The depiction of a single strand ofa nucleic acid also defines (at least partially) the sequence of thecomplementary strand. The nucleic acid may be single or double strandedor may contain portions of both double and single stranded sequences.Exemplified, double-stranded nucleic acid molecules can have 3′ or 5′overhangs and as such are not required or assumed to be completelydouble-stranded over their entire length. The nucleic acid may beobtained by biological, biochemical or chemical synthesis methods or anyof the methods known in the art, including but not limited to methods ofamplification, and reverse transcription of RNA. The term nucleic acidcomprises chromosomes or chromosomal segments, vectors (e.g., expressionvectors), expression cassettes, naked DNA or RNA polymer, primers,probes, cDNA, genomic DNA, recombinant DNA, cRNA, mRNA, tRNA, microRNA(miRNA) or small interfering RNA (siRNA). A nucleic acid can be, e.g.,single-stranded, double-stranded, or triple-stranded and is not limitedto any particular length. Unless otherwise indicated, a particularnucleic acid sequence comprises or encodes complementary sequences, inaddition to any sequence explicitly indicated.

The term “vector” refers in the context of this invention to apolynucleotide that encodes a protein of interest or a mixturecomprising polypeptide(s) and a polynucleotide that encodes a protein ofinterest, which is capable of being introduced or of introducingproteins and/or nucleic acids comprised therein into a cell. Examples ofvectors include but are not limited to plasmids, cosmids, phages,viruses or artificial chromosomes. A vector is used to introduce a geneproduct of interest, such as e.g. foreign or heterologous DNA into ahost cell. Vectors may contain “replicon” polynucleotide sequences thatfacilitate the autonomous replication of the vector in a host cell.Foreign DNA is defined as heterologous DNA, which is DNA not naturallyfound in the host cell, which, for example, replicates the vectormolecule, encodes a selectable or screenable marker, or encodes atransgene. Once in the host cell, the vector can replicate independentlyof or coincidental with the host chromosomal DNA, and several copies ofthe vector and its inserted DNA can be generated. In addition, thevector can also contain the necessary elements that permit transcriptionof the inserted DNA into an mRNA molecule or otherwise cause replicationof the inserted DNA into multiple copies of RNA. Vectors may furtherencompass “expression control sequences” that regulate the expression ofthe gene of interest. Typically, expression control sequences arepolypeptides or polynucleotides such as promoters, enhancers, silencers,insulators, or repressors. In a vector comprising more than onepolynucleotide encoding for one or more gene products of interest, theexpression may be controlled together or separately by one or moreexpression control sequences. More specifically, each polynucleotidecomprised on the vector may be control by a separate expression controlsequence or all polynucleotides comprised on the vector may becontrolled by a single expression control sequence. Polynucleotidescomprised on a single vector controlled by a single expression controlsequence may form an open reading frame. Some expression vectorsadditionally contain sequence elements adjacent to the inserted DNA thatincrease the half-life of the expressed mRNA and/or allow translation ofthe mRNA into a protein molecule. Many molecules of mRNA and polypeptideencoded by the inserted DNA can thus be rapidly synthesized. Suchvectors may comprise regulatory elements, such as a promoter, enhancer,terminator and the like, to cause or direct expression of saidpolypeptide upon administration to a subject. Examples of promoters andenhancers used in the expression vector for animal cell include earlypromoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR promoterand enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987),promoter (Mason J O et al. 1985) and enhancer (Gillies S D et al. 1983)of immunoglobulin H chain and the like. Any expression vector for animalcell can be used, as long as a gene encoding the human antibody C regioncan be inserted and expressed. Examples of suitable vectors includepAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987),pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSG1 betad2-4-(Miyaji H et al. 1990) and the like. Other examples of plasmidsinclude replicating plasmids comprising an origin of replication, orintegrative plasmids, e.g. pUC, pcDNA, pBR.

The term “antigen binding part” or “or antigen binding fragment” in thecontext of the present invention refers to molecules, in particularamino acid chains, that are shorter in length but which retain thebinding specificity and/or selectivity of the parent protein because itstill comprises the essential amino acid sequence or sequences which areresponsible for the binding specificity and/or selectivity of the parentprotein. An “antigen binding part” or “or antigen binding fragment” isconsidered to have retained the binding specificity, if it's K_(d) tothe target of the parent protein measured as outlined below is at least10-fold higher or less, 5-fold higher or less, 3-fold higher or less2-fold higher or less or identical to the K_(d) of the parent protein.Antigen binding fragments of TCRs are, e.g. the variable domains of thealpha and beta chain, and antigen binding parts of antibodies are thevariable light and heavy chain. Antigen binding parts of a TCR, BCR oran antibody are the CDRs that are positioned in the respective variableregions of the alpha and beta or light and heavy chain. Thus, if theAssessment of binding and/or specificity of an antigen binding protein,e.g., an antibody, TCR, BCR or immunologically functional part orfragment thereof, can be conducted by binding affinity measurements ofe.g. a TCR to its target peptide or an antibody to its antigen. The term“fragment” used herein refers to naturally occurring fragments (e.g.splice variants or peptide fragments) as well as artificiallyconstructed fragments, in particular to those obtained bygene-technological means.

The term “K_(d)” (measured in “mol/L”, sometimes abbreviated as “M”) inthe context of the present invention refers to the dissociationequilibrium constant of the particular interaction between a bindingmoiety (e.g. an antibody or fragment thereof) and a target molecule(e.g. an antigen or epitope thereof). Affinity can be measured by commonmethods known in the art, including but not limited to surface plasmonresonance (SPR) based assay (such as the BIAcore assay); biolayerinterferometry (BLI), enzyme-linked immunoabsorbent assay (ELISA); andcompetition assays (e.g. radio immuno assays (RIA)). Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for the purposesof the present invention. The IQ's indicated for various antigen bindingproteins throughout this disclosure are measured at room temperature,i.e. 20° C., by SPR.

The term “B-cell receptor” (BCR) refers to a receptor with an antigenlike structure present on the surface of B cells. A B-cell is activatedby its first encounter with an antigen that binds to its receptor (its“cognate antigen”), the cell proliferates and differentiates to generatea population of antibody-secreting plasma B-cells and memory B-cells.The BCR controls B-cell activation by biochemical signaling and byphysical acquisition of antigens from immune synapses withantigen-presenting cells and has two crucial functions upon interactionwith the antigen. One function is signal transduction, involving changesin receptor oligomerization. The second function is to mediateinternalization for subsequent processing of the antigen andpresentation of peptides to helper T-cells. The portion of the BCR thatrecognizes antigens is made up of three disparate genetic regions,termed V, D, and J, that are spliced and recombined at the genetic levelin a combinatorial process unique to the immune system. Theimmunoglobulin molecules that form a type 1 transmembrane receptorprotein are usually located on the outer surface of a B-lymphocyte.Structurally, the BCR comprises a membrane-bound immunoglobulin moleculeof one isotype (IgD, IgM, IgA, IgG, or IgE) and a signal transductionmoiety: A Ig-α/Ig-β (CD79) heterodimer, linked by disulfide bridges.Each member of the dimer spans the plasma membrane and has a cytoplasmictail bearing an immunoreceptor tyrosine-based activation motif (ITAM).

The term “antibody” in the context of the present invention refers tosecreted immunoglobulins which lack the transmembrane region and canthus, be released into the bloodstream and body cavities. Humanantibodies are grouped into different isotypes based on the heavy chainthey possess. There are five types of human Ig heavy chains denoted bythe Greek letters: α, γ, δ, ε, and μ. The type of heavy chain presentdefines the class of antibody, i.e. these chains are found in IgA, IgD,IgE, IgG, and IgM antibodies, respectively, each performing differentroles, and directing the appropriate immune response against differenttypes of antigens. Distinct heavy chains differ in size and composition;and may comprise approximately 450 amino acids (Janeway et al. (2001)Immunobiology, Garland Science). IgA is found in mucosal areas, such asthe gut, respiratory tract and urogenital tract, as well as in saliva,tears, and breast milk and prevents colonization by pathogens (Underdown& Schiff (1986) Annu. Rev. Immunol. 4:389-417). IgD mainly functions asan antigen receptor on B-cells that have not been exposed to antigensand is involved in activating basophils and masT-cells to produceantimicrobial factors (Geisberger et al. (2006) Immunology 118:429-437;Chen et al. (2009) Nat. Immunol. 10:889-898). IgE is involved inallergic reactions via its binding to allergens triggering the releaseof histamine from masT-cells and basophils. IgE is also involved inprotecting against parasitic worms (Pier et al. (2004) Immunology,Infection, and Immunity, ASM Press). IgG provides the majority ofantibody-based immunity against invading pathogens and is the onlyantibody isotype capable of crossing the placenta to give passiveimmunity to fetus (Pier et al. (2004) Immunology, Infection, andImmunity, ASM Press). In humans there are four different IgG subclasses(IgG1, 2, 3, and 4), named in order of their abundance in serum withIgG1 being the most abundant (66%), followed by IgG2 (23%), IgG3 (˜7%)and IgG (˜4%). The biological profile of the different IgG classes isdetermined by the structure of the respective hinge region. IgM isexpressed on the surface of B-cells in a monomeric form and in asecreted pentameric form with very high avidity. IgM is involved ineliminating pathogens in the early stages of B-cell mediated (humoral)immunity before sufficient IgG is produced (Geisberger et al. (2006)Immunology 118:429-437). Antibodies are not only found as monomers butare also known to form dimers of two Ig units (e.g. IgA), tetramers offour Ig units (e.g. IgM of teleost fish), or pentamers of five Ig units(e.g. mammalian IgM). Antibodies are typically made of four polypeptidechains comprising two identical heavy chains and identical two lightchains which are connected via disulfide bonds and resemble a “Y”-shapedmacro-molecule. Each of the chains comprises a number of immunoglobulindomains out of which some are constant domains and others are variabledomains Immunoglobulin domains consist of a 2-layer sandwich of between7 and 9 antiparallel ˜-strands arranged in two ˜-sheets. Typically, theheavy chain of an antibody comprises four Ig domains with three of thembeing constant (CH domains: CHI. CH2. CH3) domains and one of the beinga variable domain (VH). The light chain typically comprises one constantIg domain (CL) and one variable Ig domain (V L). The VH and VL regionscan be further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four 1-Rs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (Clq) of the classicalcomplement system. Term antibody as used herein also encompasses achimeric antibody, a humanized antibody or a human antibody.

The term “antigen-binding fragment” of an antibody, TCR, or BCR or CAR(or “binding portion” or “fragment”), as used herein, refers to one ormore fragments of an antibody TCR, BCR or CAR that retain the ability tospecifically bind to an antigen. It has been shown that theantigen-binding function of an antibody, of a TCR, of a BCR or CAR canbe performed by fragments of a full-length antibody, TCR, BCR or CAR.Examples of binding fragments of antibodies encompassed within the term“antigen-binding portion of an antibody, BCR or CAR “include (i) Fabfragments, monovalent fragments consisting of the VL, VH, CL and CHdomains; (ii) F(ab′)₂ fragments, bivalent fragments comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) Fdfragments consisting of the VH and CH domains; (iv) Fv fragmentsconsisting of the VL and VH domains of a single arm of an antibody, (v)dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consistof a VH domain; (vi) isolated complementarity determining regions (CDR),and (vii) combinations of two or more isolated CDRs which may optionallybe joined by a synthetic linker. Furthermore, although the two domainsof the Fv fragment, VL and VH, are coded for by separate genes, they canbe joined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodiesare also intended to be encompassed within the term “antigen-bindingfragment” of an antibody, BCR or CAR. The term “antigen binding portionof a TCR” comprises at least CDR1 and CDR3 of the alpha and beta chainof a TCR, preferably CDR1, CDR2 and CDR3 of the alpha and beta chain.While these CDRs are preferably comprised in the context of theirnatural framework regions, they may also be comprised in anotherprotein—a so called protein scaffold—that positions them to each otherin a similar way as they are positioned in an alpha and/or beta chain.The antigen binding portion of a TCR comprises preferably the variabledomain of the alpha and beta chain. The antigen binding fragments ofantibodies, TCRs, BCRs or CARs can be included in a monomeric, dimeric,trimeric, tetrameric or multimeric protein complex to provide suchcomplex with one or more different antigen binding specificities.Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci.USA 85: 5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding fragment of an antibody”.Further formats in which antigen binding fragments of an antibody areused to create monovalent, bivalent or multivalent binding molecules areknown in the art and are termed diabody, a tetrabody, and a nanobody.Similarly, to scFV's single chain TCRs comprise the variable domains ofalpha and beta chain on one protein chain linked by a linker.

As used in this specification the term “subject” relates to an“individual”, “subject”, or “patient” which are used interchangeablyherein and refer to any mammal that may benefit from the presentinvention. In particular, the “individual” is a human being. The subjectcan be a healthy subject.

The term “subject in need thereof” in the context of this inventionrefers to a subject that suffers or is at risk of suffering a disease,for example a proliferative disease or disorder, a disease caused by avirus or a disease caused by bacteria. Such a proliferative disease ordisorder, for example cancer, involve the unregulated and/orinappropriate proliferation of cells. The proliferative disorder ordisease may be, for example, a tumor disease characterized by theexpression of the TAA, more particular of the TAA, in a cancer or tumorcell of said tumor disease.

Accordingly, a particularly preferred cancer is a TA positive cancer, inparticular a TAA positive cancer.

Abbreviations of frequently used terms throughout the claims andspecification of the present invention:

TABLE 5 Abbreviations of frequently used terms. Antigen complex ACB-cell receptor BCR Chimeric antigen receptor CAR Complementarydetermining regions CDR Fluorescence activated cell sorting FACS Humanleukocyte antigen HLA Irrelevant antigen complex IAC Irrelevant peptideIP Irrelevant protein antigen IPA Magnetic activated cell sorting MACSMajor histocompatibility complex I/II MHC I/II Protein antigen ofinterest PAI Similar protein antigen SPA T-cell receptor TCR Targetpeptide TP Target similar peptide TSP Tumor-associated antigen TAA

Embodiments

In the following different aspects of the invention are defined in moredetail. Each aspect so defined may be combined with any other aspect oraspects unless clearly indicated to the contrary. In particular, anyfeature indicated as being preferred or advantageous may be combinedwith any other feature or features indicated as being preferred oradvantageous.

Immunotherapy constitutes an exciting and rapidly evolving field, andthe demonstration that genetically modified T-cell receptors (TCRs) canbe used to produce T-lymphocyte populations of desired specificityoffers new opportunities for antigen-specific T-cell therapy.

Overall, TCR-modified T-cells have the ability to target a wide varietyof self and non-self-targets through the normal biology of a T-cell.However, “off-tumor/on-target” or “off-tumor/off-target” effects canlead to tremendously undesired effects of immune related toxicity. Byincluding similar protein antigens (similar peptides) already at thestage of identification of TCRs, the inventors are able to exclude aproportion of cross-reactive T-cells before the characterization processand by that enhancing the efficiency of the whole TCR discoveryprocedure. For that purpose, 1D-labeled or 2D-labeled similar-peptidemultimers are included to the staining panel. An in-house databaseallowed the inventors to identify highly relevant, target-sequencesimilar peptides found on normal human tissue. Such target-sequencesimilar peptides pose a safety risk if recognized by a TCR that issupposed to be developed towards clinical use. Therefore, the inventorshave developed an in-house search algorithm that combines public and inhouse genomic database searches for target-similar peptides with resultsof actual MS-detected peptides on healthy tissue from the in-housedatabase. The inventors use a set of target-similar peptides earlyduring TCR identification, enabling early de-selection of cross-reactiveTCRs. For this purpose, fluorochrome (streptavidin) labelled peptidemajor histocompatibility complex (pMHC) tetramers are generated both forthe target peptide as well as for target-similar peptides,distinguishable by at least one different fluorochrome. Cells positivefor both the target as well as the similar peptide are excluded fromT-cell sorting for downstream TCR identification.

This surprising finding provides inter alia the following advantagesover the art: (i) reduction of cross-reactivity of selected TCRs withsimilar peptides on healthy tissues (ii) increased safety profile ofselected TCRs; (iii) efficient and fast identification andcharacterization of TCRs due to early stage selection of target specificTCRs; (v) specific TCR selection by exclusion of similar peptide bindingduring sorting (vi) TCRs exerting reduced off target and off-tumorcytotoxicity; and (vii) the provision of improved specific, selectiveand safe TCRs.

A first aspect of the invention relates to a method for selecting a cellor a virus expressing on its surface an antigen-binding proteinspecifically and/or selectively binding to a protein antigen of interest(PAI) comprising the following steps:

-   (i) providing a cell population or a virus population;-   (ii) contacting the cell population or the virus population of    step (i) with a first antigen complex (1^(st) AC) comprising the PAI    and a detectable label A or with the PAI comprising a detectable    label A;-   (iii) contacting the cell population or the virus population of    step (i) with at least a second antigen complex (2^(nd) AC)    comprising a similar protein antigen (SPA), wherein the amino acid    sequence of the SPA differs by at least 1 amino acid from the amino    acid sequence of the PAI and wherein the 2^(nd) AC comprises a    detectable label B; or with the SPA and a detectable label B; and-   (iv) selecting at least cell or a virus that specifically and/or    selectively binds to the 1^(st) AC,    wherein the detectable label A and the detectable label B are    detectably different from each other.

In one embodiment of the first aspect of the invention a cell isselected based on the principle of counterselection: MHC-presented shortpeptides of tumor antigens (TP) that are preferably expressed ondiseased tissue are labeled with a detectable label and peptides with asimilar sequence (TSP) that are expressed on healthy tissues are labeledwith a detectable label. The labels used in this approach are detectablydifferent. Upon contacting a cell, preferably a T-cell, with the TP andthe TSP, the cell binds to either the TP, the TSP or to both the TP andthe TSP or none and is either selected based on a positive selectioncriterion or on a negative selection criterion. In a conventionalsorting approach, the following cells with their respective detectablecell signal can be identified: One cell can be detected by detecting thesignal of the TP's label, i.e. the peptide of interest in case a cell isbound to the MHC presented peptide. Another cell can signal by thedetection of the TP's label, i.e. the peptide of interest in case animmune cell is bound to the MHC presented peptide and by the detectionof the TSP's label. A third cell can solely signal by the detection ofthe TSP's label. Positively selected are only those cells which aredetectable by the TP's label because, in case the cell is a T-cell, thisis the cell with a TCR of interest capable of binding to theMHC-presented peptides. The counterselection (or negative selection) canbe described as follows: Cells detected by two labels, i.e. the label ofTP and the TSP are negatively selected because these cells bind to theTSP beside binding to the TP. Cells only detected by the TSP's label arealso negatively selected as they do only bind the TSP which is expressedon healthy tissue and generally, binding to TSP should be avoided inorder to decrease off target effects. Often, the binding of a given cellto a given PAI, preferably a TP and one or more SPAs, preferably TSPs,is not all or nothing. Thus, the selection may also be based on relativedifferences of the binding of the PAI, preferably a TP, and a SPA,preferably a TSP. Cells are considered to specifically bind to a PAI,preferably a TP, if their binding is at least 2-fold, at least 3-fold,at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, atleast 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, atleast 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, atleast 70-fold, at least 100-fold, at least 200-fold stronger atidentical concentration of the PAI and the SPA. Preferably, the bindingis at least 10-fold stronger to the PAI, preferably the TP, than to anyof the one or more SPAs, preferably TSPs used in the method of theinvention. It is even more preferably, that the binding is at least20-fold, more preferably at least 50-fold stronger. The strength of thebinding or affinity of a given cell, for example a T-cell or its TCR, ora B-cell can be determined by a variety of assays and is commonlyindicated as the dissociation constant (K_(d)) of the TCR. However, forthe purpose of the method of the present invention an exactdetermination of the K_(d) of the cell to a given PAI, preferably a TPand one or more SPAs, preferably TSPs, is not required.

It is sufficient to determine relative binding strength, which can be,for example, determined by FACS analysis of cells in which thefluorescence signal of a given TP with the fluorescence signal of one ormore TSPs is compared. In such a determination the relative molaramounts of the PAI, preferably the TP, and of the one or more SPA,preferably TSPs, has to be taken into consideration. If TP and TSPs areadded to the cells at the same molar amounts and differences influorescent intensity of the respective labels used are accounted for, acell specifically binds to a PAI, preferably TP, if an equimolar basisof the SPA, preferably TSP, shows at least 10-fold stronger fluorescenceattributable to the PAI, preferably to the TP, than to the SPA,preferably TSP. The skilled person in the art understands that a changeof the molar ratios of PAI, preferably TP, and the SPAs, preferablyTSPs, which are contacted with the cell population in steps (ii) and(iii) can be accounted for when selecting the cells by adapting thegating accordingly.

In one embodiment of the first aspect of the present invention theselected cell is a mammalian cell that expresses a heterologous antigenbinding protein or a yeast cell that expresses a heterologous antigenbinding protein. The mammalian cell can be any mammalian cell, such as ahuman cell, a mouse cell, preferably a humanized mouse cell, a rat cell,a pig cell, a monkey cell or a dog cell. Typically, the mammalian cellcan be any antigen presenting cell (APC). Preferably, the mammalian cellis a human cell. In particular, the mammalian cell is engineered toexpress a heterologous antigen binding protein, such as a TCR orfragments thereof, or a BCR or fragments thereof or a CAR or fragmentsthereof or an antibody or fragments thereof. If the selected cell is ayeast cell expressing a heterologous antigen binding protein, it ispreferred that such a yeast cell is a Saccharomyces cerevisiae yeastcell. In particular, the yeast cell is engineered to express aheterologous antigen binding protein, such as a TCR or fragmentsthereof, or a BCR or fragments thereof or a CAR or fragments thereof oran antibody or fragments thereof.

In another embodiment of the first aspect of the present invention themethod selects a virus. The selected virus can be any virus selectedfrom the group consisting of a double-stranded DNA virus, preferablyMyoviridae, Siphoviridae, Podoviridae, Herpesviridae, Adenoviridae,Baculoviridae, Papillomaviridae, Polydnaviridae, Polyomaviridae,Poxviridae; a single-stranded DNA virus, preferably Anelloviridae,Inoviridae, Parvoviridae; double-stranded RNA virus, preferablyReoviridae; a single-stranded RNA virus, preferably Coronaviridae,Picornaviridae, Caliciviridae, Togaviridae, Flaviviridae, Astroviridae,Arteriviridae, Hepeviridae; negative-sense single-stranded RNA virus,preferably Arenaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae,Bunyaviridae, Orthomyxoviridae, Bornaviridae; a single-stranded RNAreverse transcribing virus, preferably Retroviridae; or adouble-stranded RNA reverse transcribing virus, preferablyCaulimoviridae, Hepadnaviridae. More preferably, the selected virus is abacteriophage. The bacteriophage is preferably selected from the groupconsisting of bacteriophage T4 lambda (T4λ) phage, T7 phage, fdfilamentous phage, preferably filamentous phage M13. A selected virus,for example a phage, can be bound to beads, for example magnetic beadswhich are suitable for sequential magnetic sorting. In this embodiment,it is preferred that labels, such as label A and label B are barcodelabels, preferably RNA-barcodes or DNA-barcodes as described hereinabove.

In another embodiment of the first aspect of the present invention themethod for selecting a cell comprises in step (i) providing a cellpopulation. Preferably, the cell population comprises eukaryotic cells.More preferably eukaryotic cells are mammalian cells expressing alibrary of heterologous antigen binding proteins or yeast cellsexpressing a library of heterologous antigen binding proteins. Themethod of the first aspect of the present invention can, thus, be usedfor example in a yeast display.

In another embodiment of the first aspect of the invention the methodfor selecting a virus comprises in step (i) providing a viruspopulation. Preferably, the virus population comprises virusesexpressing a library of heterologous antigen binding proteins. In apreferred embodiment the virus population comprises bacteriophages andthus, the method of the first aspect of the present invention can beused, for example, in a phage display.

Another embodiment of the first aspect the present invention relates toa method for selecting an immune cell expressing on its surface anantigen-binding protein specifically and/or selectively binding to aprotein antigen of interest (PAI) comprising the following steps:

-   (i) providing a cell population comprising immune cells;-   (ii) contacting the cell population of step (i) with a first antigen    complex (1^(st) AC) comprising the PAI and a detectable label A or    with the PAI comprising a detectable label A;-   (iii) contacting the cell population of step (i) with at least a    second antigen complex (2^(nd) AC) comprising a similar protein    antigen (SPA), wherein the amino acid sequence of the SPA differs by    at least 1 amino acid from the amino acid sequence of the PAI and    wherein the 2^(nd) AC comprises a detectable label B; or with the    SPA and a detectable label B; and-   (iv) selecting at least one immune cell that specifically and/or    selectively binds to the 1^(st) AC, wherein the detectable label A    and the detectable label B are detectably different from each other.

In one embodiment of the first aspect of the invention an immune cell isselected based on the principle of counterselection: MHC-presented shortpeptides of tumor antigens (TP) that are preferably expressed ondiseased tissue are labeled with a detectable label and peptides with asimilar sequence (TSP) that are expressed on healthy tissues are labeledwith a detectable label. The labels used in this approach are detectablydifferent. Upon contacting an immune cell, preferably a T-cell, with theTP and the TSP, the immune cell binds to either the TP, the TSP or toboth the TP and the TSP or none and is either selected based on apositive selection criterion or on a negative selection criterion. In aconventional sorting approach, the following cells with their respectivedetectable cell signal can be identified: One cell can be detected bydetecting the signal of the TP's label, i.e. the peptide of interest incase an immune cell is bound to the MHC presented peptide. Another cellcan signal by the detection of the TP's label, i.e. the peptide ofinterest in case an immune cell is bound to the MHC presented peptideand by the detection of the TSP's label. A third cell can solely signalby the detection of the TSP's label. Positively selected are only thosecells which are detectable by the TP's label because, in case the immunecell is a T-cell, this is the cell with a TCR of interest capable ofbinding to the MHC-presented peptides. The counterselection (or negativeselection) can be described as follows: Cells detected by two labels,i.e. the label of TP and the TSP are negatively selected because thesecells bind to the TSP beside binding to the TP. Cells only detected bythe TSP's label are also negatively selected as they do only bind theTSP which is expressed on healthy tissue and generally, binding to TSPshould be avoided in order to decrease off target effects. Often, thebinding of a given immune cell to a given PAI, preferably a TP and oneor more SPAs, preferably TSPs, is not all or nothing. Thus, theselection may also be based on relative differences of the binding ofthe PAI, preferably a TP, and a SPA, preferably a TSP Immune cells areconsidered to specifically bind to a PAI, preferably a TP, if theirbinding is at least 2-fold, at least 3-fold, at least 4-fold, at least5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least30-fold, at least 40-fold, at least 50-fold, at least 70-fold, at least100-fold, at least 200-fold stronger at identical concentration of thePAI and the SPA. Preferably, the binding is at least 10-fold stronger tothe PAI, preferably the TP, than to any of the one or more SPAs,preferably TSPs used in the method of the invention. It is even morepreferably, that the binding is at least 20-fold, more preferably atleast 50-fold stronger. The strength of the binding or affinity of agiven immune cell, e.g. a T-cell or its TCR can be determined by avariety of assays and is commonly indicated as the dissociation constant(K_(d)) of the TCR. However, for the purpose of the method of thepresent invention an exact determination of the K_(d) of the immune cellto a given PAI, preferably a TP and one or more SPAs, preferably TSPs,is not required. It is sufficient to determine relative bindingstrength, which can be, for example, determined by FACS analysis ofimmune cells in which the fluorescence signal of a given TP with thefluorescence signal of one or more TSPs is compared. In such adetermination the relative molar amounts of the PAI, preferably the TP,and of the one or more SPA, preferably TSPs, has to be taken intoconsideration. If TP and TSPs are added to the immune cells at the samemolar amounts and differences in fluorescent intensity of the respectivelabels used are accounted for, an immune cell specifically binds to aPAI, preferably TP, if an equimolar basis of the SPA, preferably TSP,shows at least 10-fold stronger fluorescence attributable to the PAI,preferably to the TP, than to the SPA, preferably TSP. The skilledperson in the art understands that a change of the molar ratios of PAI,preferably TP, and the SPAs, preferably TSPs, which are contacted withthe cell population in steps (ii) and (iii) can be accounted for whenselecting the cells by adapting the gating accordingly.

In one embodiment the cell population of step (i) is contacted with atleast a second antigen complex (2^(nd) AC) comprising a SPA, preferablya TSP, at least a third antigen complex (3^(rd) AC) comprising a SPA,preferably a TSP, at least a fourth antigen complex (4^(th) AC)comprising a SPA, preferably a TSP, at least a fifth antigen complex(5^(th) AC) comprising a SPA, preferably a TSP, at least a sixth antigencomplex (6^(th) AC) comprising a SPA, preferably a TSP, at least aseventh antigen complex (7^(th) AC) comprising a SPA, preferably a TSP,at least a eighth antigen complex (8^(th) AC) comprising a SPA,preferably a TSP, at least a ninth antigen complex (9^(th) AC)comprising a SPA, preferably a TSP, at least a fifth antigen complex(10^(th) AC) comprising a SPA, preferably a TSP. Accordingly, it ispreferred that not more than ten SPAs, preferably TSPs, are used, notmore than nine SPAs, preferably TSPs are used, not more than eight SPAs,preferably TSPs, are used, not more than seven SPAs, preferably TSPs areused, not more than six SPAs, preferably TSPs are used, not more thanfive SPAs, preferably TSPs are used, not more than four SPAs, preferablyTSPs, are used, not more than three SPAs, preferably TSPs are used, notmore than two SPAs, preferably TSPs, are used or not more than one SPA,preferably one TSP is used. Accordingly, it is preferred that in themethod of the present invention between 1-10 different TSPs are used,preferably 2-8 different TSPs and more preferably 3-5 different TSPs. Inan even more preferred embodiment, three TSPs are used.

In one embodiment the method of the first aspect of the inventionselects an immune cell. Preferably this immune cell is a T-cell or aB-cell. More preferably, the T-cell is a CD4 T-cell. In an even morepreferred embodiment the T-cell is a CD8 T-cell. The signaling domainpreferably comprises CD3. In another embodiment of the first aspect ofthe invention the immune cell expresses on its surface an antigenbinding protein. It is preferred that the antigen binding protein is aTCR or an antigen binding fragment thereof if the immune cell to beselected is a T-cell. It is preferred that the antigen binding proteinis a BCR or an antigen binding fragment thereof if the immune cell to beselected is a B-cell. Such an antigen recognition or antigen bindingsite is preferably a single chain variable fragment (scFv) andpreferably targets a PAI that is a TAA. The costimulatory domainpreferably comprises CD28 or 4-1 BB. The signaling domain preferablycomprises CD3. It is also preferred that the antigen binding protein isa CAR or an antigen binding fragment thereof if the immune cell to beselected is a T-cell.

In another embodiment of the first aspect of the invention the methodfor selecting an immune cell comprises in step (i) providing a cellpopulation comprising immune cells. The cell population comprisingimmune cells is derived from peripheral blood of healthy subjects. Inanother embodiment, the cell population comprising immune cells isderived from peripheral blood from diseased subjects. Preferably thecell population comprising immune cells is derived from an immune cellenriched fraction of the peripheral blood of a healthy or diseasedsubject. Preferably, the immune cell enriched fraction is enriched instem cells, T-cells, B-cells or plasma cells. It is even more preferredthat the immune enriched fraction is enriched in CD4 T-cells and/or CD8T-cells. In another embodiment the cell population comprising immunecells can be derived from tumor-infiltrating lymphocytes (TILs) or TCRlibraries. Preferably, the TCR library contains a high number ofdifferent T cell receptor (TCR) proteins or fragments thereof, whereineach TCR protein or fragment thereof is different.

In another embodiment of the first aspect of the invention the immunecell or cells in the immune cell enriched fraction are selected bydetectably labeling one or more immune cell specific surface markers. Itis preferred that the immune cell surface markers are selected from thegroup consisting of CD3, CD8, CD4 and CD19.

In another embodiment of the first aspect of the invention, the cellpopulation of step (i) a can be incubated in the presence of growthfactors and/or cytokines in a further step. Preferably, cytokines areinterleukins. More preferably, interleukins are selected from the groupconsisting of IL-1, IL-2, IL-7, IL-10, IL-12, Il-15, IL-17, IL-21 andIL-23. Most preferably, the cell population of step (i) is incubatedwith IL-2, IL-7, IL-15 and/or IL-21.

In another embodiment the protein antigen of interest (PAI) is a tumorassociated antigen (TAA), a viral protein or a bacterial protein. It ispreferred that if the PAI is a target peptide, i.e. a shorter fragmentof the PAI, the target peptide is a viral antigenic peptide or abacterial antigenic peptide. In another embodiment of the first aspectof the invention the diseased subject suffers from a disease selectedfrom the group consisting of an immune disease, a neoplastic disease, adisease cause by a virus or a disease caused by bacteria. Preferably theneoplastic disease is cancer. Preferably, diseases caused by a virus isa viral infection; and a disease caused by bacteria is a bacterialinfection. Preferably, the viral infection is caused by a virus selectedfrom the group consisting of HIV, HCMV, CMV, HPV, HBV, HCV, HPV, EBV,Influenza virus. More preferably the viral infection is caused by HIV.Preferably, the bacterial infection is caused by Mycobacteriumtuberculosis. Such a disease is tuberculosis.

In another preferred embodiment of the first aspect the method comprisesin step (ii) contacting the cell population of step (i) with a firstantigen complex (1^(st) AC) comprising the PAI and a detectable label Aor with the PAI and a detectable label A. The 1^(st) AC comprising thePAI and the label A is preferably an antigen presenting cell. In anotherpreferred embodiment the 1^(st) AC is a complex comprising a particle,the PAI and the detectable label A. More preferably, the particle is anano- or a microbead. It is also preferred that an MHC molecule islinked to a nano- or microbead. In another preferred embodiment the1^(st) AC consists of the PAI and the detectable label A. In anotherembodiment the 1^(st) AC is a complex comprising a particle, the SPA andthe detectable label B. More preferably, the particle is a nano- or amicrobead. It is also preferred that an MHC molecule is linked to anano- or microbead. In another preferred embodiment the 1^(st) ACconsists of the SPA and the detectable label B.

Alternatively, the PAI may comprising a detectable label. This is apreferred embodiment, if the PAI is an amino acid chain and the label iscovalently linked to this amino acid chain. Examples are fluorescentlabels or fluorescent proteins as GFP or EGFP. In the latter case it ispreferred that the fluorescent proteins are linked to the PAI by apeptide bond.

In another preferred embodiment the method of the first aspect of theinvention further comprises one or more of the steps of contacting thecell population with a further AC comprising a further PAI and a furtherdetectable label and a further AC comprising a further SPA and a furtherdetectable label. Using a third AC comprising a further PAI and afurther detectable label C and a fourth AC comprising a further SPA anda further detectable label D mirrors the so called 2D Multimermultiplexing (2DMM) approach which allows specific rare cell detectionwith high sensitivity (0.0001%) in a highly cell saving manner Twomultimers labeled with different fluorochromes for each specificity(peptide MHC) enable the identification of up to 36 differentspecificities in one sample by using 9 different fluorochromes. In onepreferred embodiment the cell population of step (i) is contacted with athird antigen complex (3^(rd) AC) comprising the PAI and a detectablelabel C that is detectably different from one or more or all of theother detectable labels of the other ACs contacted with the cellpopulation. Preferably the label is detectably different from at leastthe detectable label A, preferably from at least the detectable label Aand a detectable label D, if a detectable label D is present. Adetectable label D is present if the cell population of step (i) iscontacted with a fourth antigen complex (4^(th) AC) comprising the PAIand a detectable label D that is detectably different from one or moreor all of the other detectable labels of the other ACs contacted withthe cell population. The detectable label D is preferably detectablydifferent from at least the detectable label A. It is also preferredthat the detectable label D is detectably different from at least thedetectable label A and the detectable label C. In one embodiment thecell population of step (i) is contacted with a fifth antigen complex(5^(th) AC) comprising the SPA and a detectable label E that isdetectably different from one or more or all of the other detectablelabels of the other ACs contacted with the cell population. It ispreferred that the label E is detectably different from at least thedetectable label B. It is also preferred that the detectable label E isdetectably different from at least the detectable label B and adetectable label F, (6) if a detectable label F is present. A detectablelabel F is present if the cell population of step (i) is furthercontacted with a sixth antigen complex (6^(th) AC) comprising the SPAand a detectable label F that is detectably different from one or moreor all of the other detectable labels of the other ACs contacted withthe cell population. It is preferred that the label F is detectablydifferent from at least the detectable label B. It is also preferredthat the detectable label F is detectably different from at least thedetectable label B and the detectable label E.

In another embodiment the cell population of step (i) is contacted witha first antigen complex (1^(st) AC) comprising the PAI, preferably a TP,and a detectable label A; and with at least a second antigen complex(2^(nd) AC) comprising a similar protein antigen (SPA), preferably aTSP, wherein the amino acid sequence of the SPA, preferably the TSP,differs by at least 1 amino acid from the amino acid sequence of thePAI, preferably the TP, and wherein the 2^(nd) AC comprises a detectablelabel B which is detectably different to label A; and with one to ten,i.e. one, two, three, four, five, six, seven, eight, nine or ten,preferably two to four, most preferably two further antigen complexes(ACs), wherein each comprises a different SPA, preferably a differentTSP, that differs in at least one amino acid sequence from the aminoacid sequence of the SPA of the 2^(nd) AC, and wherein each further ACcomprises one or more labels, wherein the one or more label isdetectably different to the one or more labels of the 2^(nd) AC. It ispreferred that the 1^(st) AC comprises a further, second label which isdetectably different to the one or more labels of the 2^(nd) AC and tothe one or more labels of the further ACs. It is also preferred that thecell population is a T-cell population. It is further preferred that theselected immune cell that specifically and/or selectively binds to the1^(st) AC is a T-cell. It is also preferred that the antigen-bindingprotein on the surface of the selected T-cell which is specificallyand/or selectively binding to the PAI is a TCR.

In another embodiment the cell population of step (i) is contacted witha first antigen complex (1^(st) AC) comprising the PAI, preferably a TP,and a detectable label A; and with at least a second antigen complex(2^(nd) AC) comprising a similar protein antigen (SPA), preferably aTSP, wherein the amino acid sequence of the SPA, preferably the TSP,differs by at least 1 amino acid from the amino acid sequence of thePAI, preferably the TP, and wherein the 2^(nd) AC comprises a detectablelabel B; and with one to ten, i.e. one, two, three, four, five, six,seven, eight, nine or ten, preferably two to four, most preferably twofurther antigen complexes (AC) wherein each comprises a different SPA,preferably a different TSP, that differs in at least one amino acidsequence from the amino acid sequence of the SPA of the 2^(nd) AC, andwherein each further AC comprises one or more labels, wherein the one ormore labels is the same as the one or more labels of the 2^(nd) AC. Itis also preferred that the cell population is a T-cell population. It isfurther preferred that the selected immune cell that specifically and/orselectively binds to the 1^(st) AC is a T-cell. It is also preferredthat the antigen-binding protein on the surface of the selected T-cellwhich is specifically and/or selectively binding to the PAI is a TCR.

In another embodiment the cell population of step (i) is contacted witha first antigen complex (1^(st) AC) comprising the PAI, preferably a TP,and a detectable label A; and with at least a second antigen complex(2^(nd) AC) comprising a similar protein antigen (SPA), preferably aTSP, wherein the amino acid sequence of the SPA, preferably the TSP,differs by at least 1 amino acid from the amino acid sequence of thePAI, preferably the TP, and wherein the 2^(nd) AC comprises a detectablelabel B which is detectably different to label A and the 1^(st) ACcomprises at least one further detectable label and the 2^(nd) ACcomprises at least one further detectable label, which are the same. Itis preferred that the cell population is a T-cell population. It isfurther preferred that the selected immune cell that specifically and/orselectively binds to the 1^(st) AC is a T-cell. It is also preferredthat the antigen-binding protein on the surface of the selected T-cellwhich is specifically and/or selectively binding to the PAI is a TCR.

In another embodiment the cell population of step (i) is contacted witha first antigen complex (1^(st) AC) comprising the PAI, preferably a TP,and a detectable label A; and with at least a second antigen complex(2^(nd) AC) comprising SPA, preferably a TSP, wherein the amino acidsequence of the SPA, preferably the TSP, differs by at least 1 aminoacid from the amino acid sequence of the PAI, preferably the TP, andwherein the 2^(nd) AC comprises a detectable label B which is detectablydifferent to label A and the 1^(st) AC comprises at least one furtherdetectable label and the 2^(nd) AC comprises at least one furtherdetectable label, which are different. It is preferred that the cellpopulation is a T-cell population. It is further preferred that theselected immune cell that specifically and/or selectively binds to the1^(st) AC is a T-cell. It is also preferred that the antigen-bindingprotein on the surface of the selected T-cell which is specificallyand/or selectively binding to the PAI is a TCR.

In another embodiment the cell population of step (i) is contacted witha first antigen complex (1^(st) AC) comprising the PAI, preferably a TP,and a detectable label A; and with at least a second antigen complex(2^(nd) AC) comprising a similar protein antigen (SPA), preferably aTSP, wherein the amino acid sequence of the SPA, preferably the TSP,differs by at least 1 amino acid from the amino acid sequence of thePAI, preferably the TP, and wherein the 2^(st) AC comprises a detectablelabel B which is detectably different to label A and the 1^(st) ACcomprises at least one further detectable label and the 2^(nd) ACcomprises at least one further detectable label, which are the same andthe cell population of step (i) is contacted with one or more furtherantigen complexes (ACs) wherein each comprises a SPA that differs in atleast one amino acid sequence from the amino acid sequence of the SPA ofthe 2^(nd) AC the one or more further AC comprises at least one furtherdetectable label; wherein the at least one further label is selected insuch that it allows to distinguish the 1^(st) AC from the 2^(nd) AC andthe one or more further ACs. It is preferred that the cell population isa T-cell population. It is further preferred that the selected immunecell that specifically and/or selectively binds to the 1^(st) AC is aT-cell. It is also preferred that the antigen-binding protein on thesurface of the selected T-cell which is specifically and/or selectivelybinding to the PAI is a TCR.

In another embodiment the cell population of step (i) is contacted witha first antigen complex (1^(st) AC) comprising the PAI, preferably a TP,and a detectable label A; and with at least a second antigen complex(2^(nd) AC) comprising a similar protein antigen (SPA), preferably aTSP, wherein the amino acid sequence of the SPA, preferably the TSP,differs by at least 1 amino acid from the amino acid sequence of thePAI, preferably the TP, and wherein the 2^(nd) AC comprises a detectablelabel B which is detectably different to label A and the 1^(st) ACcomprises at least one further detectable label and the 2^(nd) ACcomprises at least one further detectable label, which are different andthe cell population of step (i) is contacted with one or more furtherantigen complexes (ACs) wherein each comprises a SPA that differs in atleast one amino acid sequence from the amino acid sequence of the SPA ofthe 2^(nd) AC and, wherein the one or more further AC comprises at leastone further detectable label; wherein the at least one further label isselected in such that it allows to distinguish the 1^(st) AC from the2^(nd) AC and the one or more further ACs. It is preferred that the cellpopulation is a T-cell population. It is further preferred that theselected immune cell that specifically and/or selectively binds to the1^(st) AC is a T-cell. It is also preferred that the antigen-bindingprotein on the surface of the selected T-cell which is specificallyand/or selectively binding to the PAI is a TCR.

In another embodiment the cell population of step (i) is contacted witha first antigen complex (1^(st) AC) comprising the PAI, preferably a TP,and a detectable label A; and with at least a second antigen complex(2^(nd) AC) comprising a similar protein antigen (SPA), preferably aTSP, wherein the amino acid sequence of the SPA, preferably the TSP,differs by at least 1 amino acid from the amino acid sequence of thePAI, preferably the TP, and wherein the 2^(st) AC comprises a detectablelabel B which is detectably different to label A and the 1^(st) ACcomprises at least one further detectable label and the 2^(nd) ACcomprises at least one further detectable label, which are the same andthe cell population of step (i) is contacted with one to ten, i.e. one,two, three, four, five, six, seven, eight, nine or ten, preferably twoto four, more preferably two further antigen complexes (ACs) whereineach comprises a different SPA, preferably a different that differs inat least one amino acid sequence from the amino acid sequence of the SPAof the 2^(nd) AC and wherein the one or more further AC comprises atleast one further detectable label; wherein the at least one furtherlabel is selected in such that it allows to distinguish the 1^(st) ACfrom the 2^(nd) AC and the one or more further ACs. It is preferred thatthe cell population is a T-cell population. It is further preferred thatthe selected immune cell that specifically and/or selectively binds tothe 1^(st) AC is a T-cell. It is also preferred that the antigen-bindingprotein on the surface of the selected T-cell which is specificallyand/or selectively binding to the PAI is a TCR.

In each of the above embodiments it is preferred that each of the SPAs,in particular each of the TSPs has a similarity to the amino acidsequence of the PAI, in particular to the amino acid sequence of the TPof at least 50%, at least 60%, at least 70%, at least 80%, at least 90%.

In another embodiment the detectable labels are provided. The skilledperson in the art is well aware of how to label a protein antigen ofinterest, target peptide, similar protein antigen or target similarpeptide of interest. Detectable labels as specified above with A-F, areindependently selected from the group consisting of magnetic labels,fluorescent label, RNA-barcodes; DNA barcodes; or radioactive labels.Preferably, magnetic labels may comprise magnetic beads or magneticnanoparticles which can be coated with e.g. antibodies against aparticular surface antigen. Magnetic labels may be used inmagnetic-activated cell sorting (MACS). Preferably, the detectable labelis a fluorescent label selected from the group consisting of xanthens,acridines, oxazines, cyanines, styryl dyes, coumarins, porphines,metal-ligand-complexes, fluorescent proteins, nanocrystals, perylenesand phtalocyanines. Also preferred is the use of phycoerythrin (SA-PE),streptavidin-allophycocyanin (SA-APC) or streptavidin-brilliant-violet421 (SA-BV421) as fluorescent labels for the detectable labels A-F. Inanother preferred embodiment the 1^(st) AC is a complex of a MHC-I orMHC-II and the PAI, and the PAI is a target peptide (TP). It ispreferred that the TP is TAA. Additionally or alternatively, the 2nd ACis a complex of a MHC-I or MHC-II and the SPA, and the SPA is a targetsimilar peptide (TSP). In a further preferred embodiment the 1^(st) ACand the 2^(nd) AC is a soluble multimerized MHC-peptide complex.

Functional Differences and Similarities of PAI and SPA:

As noted above the PAI is preferably expressed on diseased tissues,contrary to the SPA which is preferably expressed on healthy humantissues and thus, is selected based on the expression on healthytissues. The inventors developed an in-house high-throughput technologyplatform (XPRESIDENT) including a large immunopeptidome database(comprising peptides which have been previously found to be presented onhealthy tissues. SPAs are preferably from MHC, preferably HLA typedsource, i.e. the SPAs are capable of binding to the respective MHC,preferably HLA molecule. This is required in the case the immune cell isa T-cell and the SPA is presented to the T-cell bound to an HLA moleculein order to allow the T-cell to recognize HLA presented SPAs. It isthus, preferred to select a SPA that is known to be presented on thesame HLA allotype as the PAI. Preferably, SPAs are used in the method ofthe invention that are expressed on cells of healthy tissue with morethan 10 copies per cell, preferably more than 20 copies per cell,preferably more than 50 copies per cell and even more preferably morethan 100 copies per cell. The counterselection of T-cells that arecapable of binding to such relatively abundant SPAs and at the same timeto the PAI is desired to avoid off-target/off-tumor toxicity.

Number of TSP:

TCRs of T-cells recognize a subgroup of amino acids within a given TP,i.e. the epitope of the TCR. Thus, if too many different TSP are used,it is likely that there will be no TCR that predominantly or exclusivelybinds to the TP but not to the TSP. Accordingly, it is preferred thatnot more than 10 TSPs, not more than 9 TSPs are used, not more than TSPsare used, not more than seven TSPs are used, not more than six TSPs areused, not more than five TSPs are used, not more than four TSPs areused, not more than three TSPs are used, not more than 2 TSPs are usedor not more than 1 TSP is used. Accordingly, it is preferred that in themethod of the present invention between 1-10 different TSPs, between 2-8different TSPs, between 3-5 different TSPs or between one to threedifferent TSPs are used. In a preferred embodiment three TSPs are used.The TSPs are fragments of SPAs and are selected on the basis of the samecriteria as outlined for the SPA above. Similarly, TSPs are selectedthat are strongly expressed in healthy tissue. Accordingly, the TSPs tobe included in the method of the invention are those, with high sequencesimilarity as defined above, i.e. it is preferred that each of the SPAs,in particular each of the TSPs has a similarity to the amino acidsequence of the PAI, in particular to the amino acid sequence of the TPof at least 50%, at least 60%, at least 70%, at least 80%, or at least90%, and that show the highest expression on healthy tissue.

Length of TP and TSP:

In one embodiment, the TP comprises 8-11 amino acids in length. The TPmay also comprise 12 amino acids. In one embodiment, the TP comprises13-25 amino acids in length. In another embodiment, the TP comprises13-18 amino acids in length. Typically, the TSPs are chosen to have thesame length as the given TP. However, alternatively, the length of theTSP may be longer or shorter by one to three amino acids as the TP. Inthe embodiments in which the TP is MHC presented, the length of the oneor more TSPs are chosen in such that they can also be MHC presented. Forexample, if the TP comprises 8 amino acids in length, it is preferredthat the TSP either has a length of 7 or less amino acids or a length of8 or more amino acids. More preferably, a mixture of TSPs comprisingsequences of different amino acids in length are used. In an embodimentwherein the TP is bound to MHC-I, the TP comprises 8-12 amino acids inlength. In another embodiment, wherein the TP is bound to MHC-I, the TPcomprises 8-11 amino acids in length. In an embodiment wherein the TP isbound to MHC-I, the TP comprises 8-10 amino acids in length. In anembodiment wherein the TP is bound to MHC-II, the TP comprises 13-23amino acids in length. In a preferred embodiment wherein the TP is boundto MHC-II, the TP comprises 13-18 amino acids in length.

Structural Difference/Similarity of TP and TSP:

In another embodiment the TSP is selected from the XPRESIDENT databaseof healthy tissue-presented HLA bound peptides based on high sequencesimilarity (similarity BLAST search) to the TP. The XPRESIDENT databasecomprises peptides presented by different HLA allotypes on healthy ordiseased tissues. It is preferred that the TSP and the TP are presentedby the same HLA allotype. HLA allotypes presenting TSP and TPs can beselected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F,HLA-G, HLA-H, HLA-J, HLA-K, HLA-L. Preferably the HLA-A protein isselected from the group consisting of HLA-A1, HLA-A2, HLA-A3, andHLA-A11. Preferred HLA-A alleles are HLA-A*02:01; HLA-A*01:01,HLA-A*03:01 or HLA-A*24:02. Preferred HLA-B alleles are HLA-B*07:02;HLA-B*08:01, HLA-B*15:01, HLA-B*35:01 or HLA-B*44:05.

Generally, for most of the HLA allotypes listed above, the second aminoacid (when counting from the N-terminus) and the C-terminal amino acidof a given MHC presented peptide are not comprised in the epitope ofthat peptide recognized by a TCR that specifically binds to thatpeptide.

In another embodiment the amino acid sequence of the TSP has a length of8 to 16 amino acids and the TP has a length of 8 amino acids and whereinthe amino acid sequence of the TSP differs from the amino acid sequenceof the TP as follows:

(SEQ ID NO: 1) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈

-   (i) at position X₁, X₂ and X₃, and wherein position X₄ to X₈ are    identical or similar, preferably identical to the TP; or-   (ii) at position X₄, X₅ and X₆, and wherein positions X₁ to X₃ and    X₇ and X₈ are identical or similar, preferably identical to the TP;    or-   (iii) at position X₇ and X₈, and wherein position X₁ to X₆ are    identical or similar, preferably identical to the TP.

In a preferred embodiment the positions X₁, X₂ and X₃ are mutated in theTSP wherein the position X₄ to X₈ are identical compared to the TP. Inanother preferred embodiment positions X₄, X₅ and X₆ are mutated in theTSP and positions X₁ to X₃ and X₇ and X₈ are identical to the TP. Inanother preferred embodiment position X₇ and X₈ in the TSP are mutatedand position X₁ to X₆ are identical to the TP. In another preferredembodiment position X₇ and X₈ in the TSP are mutated and position X₁ toX₆ are identical to the TP.

In another preferred embodiment a mixture of the TSP described above in(i) to (iii), i.e. TSP with different mutation patterns are used in themethod of the first aspect of the invention. In another preferredembodiments a mixture of the TSP described above in (i) to (iii), i.e.TSP with different mutation patterns and also different amino acidsequences in length are used. The use of such a mixture of TSP allowsthe fast and efficient positive selection of immune cells binding to theTP and negative selection of immune cells binding to one or more TSPs.

In another preferred embodiment the amino acid sequence of the TSP has alength of 8 to 16 amino acids and the TP has a length of 9 amino acidsand the amino acid sequence of the TSP differs from the amino acidsequence of the TP

(SEQ ID NO: 2) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉

-   (i) at position X₁, X₂ and X₃, and wherein position X₄ to X₉ are    identical or similar, preferably identical to the TP;-   (ii) at position X₄, X₅ and X₆, and wherein position X₁ to X₃ and    positions X₇ to X₉ are identical or similar, preferably identical to    the TP; or-   (iii) at position X₄, X₅, X₆ and X₇, and wherein position X₁ to X₃    and positions X₈ to X₉ are identical to the TP; or-   (iv) at position X₇, X₈ and X₉, and wherein position X₁ to X₆ are    identical or similar, preferably identical to the TP.

In a preferred embodiment the positions X₁, X₂ and X₃ are mutated in theTSP wherein the position X₄ to X₉ are identical compared to the TP. Inanother preferred embodiment positions X₄, X₅ and X₆ and X₇ are mutatedin the TSP and positions X₁ to X₃ and positions X₈ and X₉ are identicalto the TP. In another preferred embodiment positions X₇ to X₉ aremutated in the TSP and positions X₁ to X₆ are identical to the TP. Inanother preferred embodiment position X₇ to X₉ in the TSP are mutatedand position X₁ to X₆ are identical to the TP. In another preferredembodiment a mixture of the TSP described above in (i) to (iv), i.e. TSPwith different mutation patterns are used in the method of the firstaspect of the invention. In another preferred embodiments a mixture ofthe TSP described above in (i) to (iv), i.e. TSP with different mutationpatterns and also different amino acid sequences in length are used.

In another preferred embodiment the amino acid sequence of the TSP has alength of 8 to 16 amino acids, the TP has a length of 10 amino acids andthe amino acid sequence of the TSP differs from the amino acid sequenceof the TP

(SEQ ID NO: 3) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀

-   (i) at position X₁, X₂ and X₃, wherein position X₄ to X₁₀ are    identical or similar, preferably identical to the TP;-   (ii) at position X₄, X₅, X₆ and X₇, wherein position X₁ to X₃ and    positions X₈ to X₁₀ are identical or similar, preferably identical    to the TP; or-   (iii) at position X₄, X₅ and X₆, and wherein position X₁ to X₃ and    positions X₇ to X₁₀ are identical or similar, preferably identical    to the TP; or-   (iv) at position X₈, X₉ and X₁₀, wherein position X₁ to X₇ are    identical or similar, preferably identical to the TP.

In a preferred embodiment the positions X₁, X₂ and X₃ are mutated in theTSP wherein the position X₄ to X₁₀ are identical compared to the TP. Inanother preferred embodiment positions X₄ to X₇ are mutated in the TSPand positions X₁ to X₃ and positions X₈ and X₁₀ are identical to the TP.In another preferred embodiment position X₄ to X₆ are mutated in the TSPand position X₁ to X₃ and positions X₇ to X₁₀ are identical to the TP.In another preferred embodiment positions X₈ to X₁₀ are mutated in theTSP and positions X₁ to X₇ are identical to the TP. In another preferredembodiment a mixture of the TSP described above in (i) to (iv), i.e. TSPwith different mutation patterns are used in the method of the firstaspect of the invention. In another preferred embodiments a mixture ofthe TSP described above in (i) to (iv), i.e. TSP with different mutationpatterns and also different amino acid sequences in length are used.

In another preferred embodiment the amino acid sequence of the TSP has alength of 8 to 16 amino acids, the TP has a length of 11 amino acids andthe amino acid sequence of the TSP differs from the amino acid sequenceof the TP

(SEQ ID NO: 4) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X_(s)-X₉-X₁₀X₁₁

-   (i) at position X₁, X₂ and X₃, wherein position X₄ to X₁₁ are    identical or similar, preferably identical to the TP;-   (ii) at position X₄, X₅, X₆ and X₇, wherein position X₁ to X₃ and    positions X₈ to X₁₁ are identical or similar, preferably identical    to the TP; or-   (iii) at position X₄, X₅ and X₆, and wherein position X₁ to X₃ and    positions X₇ to X₁₁ are identical or similar, preferably identical    to the TP; or-   (iv) at position X₈, X₉, X₁₀ and X₁₁, wherein position X₁ to X₇ are    identical or similar, preferably identical to the TP; or-   (v) at position X₉, X₁₀ and X₁₁, wherein position X₁ to X₈ are    identical or similar, preferably identical to the TP.

In a preferred embodiment the positions X₁, X₂ and X₃ are mutated in theTSP wherein the position X₄ to X₁₁ are identical compared to the TP. Inanother preferred embodiment positions X₄ to X₇ are mutated in the TSPand positions X₁ to X₃ and positions X₈ to X₁₁ are identical to the TP.In another preferred embodiment position X₄ to X₆ are mutated in the TSPand position X₁ to X₃ and positions X₇ to X₁₁ are identical to the TP.In another preferred embodiment positions X₅ to X₁₁ are mutated in theTSP and positions X₁ to X₇ are identical to the TP. In another preferredembodiment positions X₉ to X₁₁ are mutated in the TSP and positions X₁to X₈ are identical to the TP.

In another preferred embodiment a mixture of the TSP described above in(i) to (iv), i.e. TSP with different mutation patterns are used in themethod of the first aspect of the invention. In another preferredembodiments a mixture of the TSP described above in (i) to (iv), i.e.TSP with different mutation patterns and also different amino acidsequences in length are used.

In another preferred embodiment the amino acid sequence of the TSP has alength of 8-16 amino acids, the TP has a length of 12 amino acids andthe amino acid sequence of the TSP differs from the amino acid sequenceof the TP

(SEQ ID NO: 5) X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀X₁₁X₁₂ 

-   (i) at position X₁, X₂ and X₃, wherein position X₄ to X₁₂ are    identical or similar to the TP;-   (ii) at position X₄, X₅, X₆ and X₇, wherein position X₁ to X₃ and    positions X₅ to X₁₂ are identical or similar to the TP; or-   (iii) at position X₄, X₅ and X₆, and wherein position X₁ to X₃ and    positions X₇ to X₁₂ are identical or similar to the TP; or-   (iv) at position X₈, X₉, X₁₀, X₁₁ and X₁₂, wherein position X₁ to X₇    are identical or similar to the TP; or-   (v) at position X₉, X₁₀, X₁₁ and X₁₂, wherein position X₁ to X₈ are    identical or similar to the TP.

In a preferred embodiment the positions X₁, X₂ and X₃ are mutated in theTSP wherein the position X₄ to X₁₂ are identical compared to the TP. Inanother preferred embodiment positions X₄ to X₇ are mutated in the TSPand positions X₁ to X₃ and positions X₈ to X₁₂ are identical to the TP.In another preferred embodiment position X₄ to X₆ are mutated in the TSPand position X₁ to X₃ and positions X₇ to X₁₂ are identical to the TP.In another preferred embodiment positions X₈ to X₁₂ are mutated in theTSP and positions X₁ to X₇ are identical to the TP. In another preferredembodiment positions X₉ to X₁₂ are mutated in the TSP and positions X₁to X₈ are identical to the TP.

In another preferred embodiment a mixture of the TSP described above in(i) to (iv), i.e. TSP with different mutation patterns are used in themethod of the first aspect of the invention. In another preferredembodiments a mixture of the TSP described above in (i) to (iv), i.e.TSP with different mutation patterns and also different amino acidsequences in length are used.

In another embodiment of the first aspect of the invention the aminoacid sequence of the SPA or of at least one protein or peptide comprisedin the SPA has a similarity to the amino acid sequence of the PAI of atleast 50%, at least 60%, at least 70%, at least 80%, or of at least 90%or of at least 95%. In another embodiment the amino acid sequence of theSPA or of at least one protein or peptide comprised in the SPA has lessthan or 90%, less than or 89%, less than or 88%, less than or 87% orless than or 86% amino acid identity to the PAI. In another embodimentthe amino acid sequence of the SPA or of at least one protein or peptidecomprised in the SPA has less than or 85%, less than or 84%, less thanor 83%, less than or 82%, less than or 81% or less than or 80% aminoacid identity to the PAI.

In another embodiment of the first aspect of the invention the aminoacid sequence of the TSP has less than or 96%, less than or 95%, lessthan or 94%, less than or 93%, less than or 92% or less than or 91%amino acid identity to the TP. In another embodiment the amino acidsequence of the TSP has less than 90%, less than or 89%, less than or88%, less than or 87% or less than or 86% amino acid identity to the TP.In another embodiment the amino acid sequence of the TSP has less thanor 85%, less than or 84%, less than or 83%, less than or 82%, less thanor 81% or less than or 80% amino acid identity to the TP.

In another embodiment the absolute expression of a TSP on healthy tissueis correlated with the lowest sequence identity of TSPs included in themethod of the invention. If a TSP is highly expressed on healthy tissue,TCRs which only bind with low affinity to the TSP may nevertheless bindto the TSP expressed in healthy tissue due to avidity effects. Thus, ifa given TSP has a low copy number on healthy tissue, it is included inthe method of the present invention only, if it shows a high similarityto the TP. Correspondingly, if a given TSP has a high copy number onhealthy tissue, it is included in the method of the present inventionalthough it may have a low similarity to the TP. For example, if TSPshave a copy number below 10 per healthy cell than it is included, if ithas at least a 90% sequence similarity to the TP. If the copy number ofTSPs are between 1 to 25 such TSPs are included, if they have at least85% sequence similarity with the TP. If the copy number of TSPs arebetween 25 to 100/cell such TSPs are included, if they have at least 80%sequence similarity with the TP. If the copy number of TSPs are between100 to 250/cell such TSPs are included, if they have at least 75%sequence similarity with the TP. If the copy number of TSPs are above250/cell such TSPs are included, if they have at least 50% sequencesimilarity with the TP.

In another embodiment of the method of the first aspect of the presentinvention the steps (ii) and (iii) of the method are carried outconsecutively or concomitantly. In another embodiment of the firstaspect of the invention steps (a), (b), (c) and (d) as outline above arecarried out consecutively or concomitantly. Whether steps (a), (b), (c)and (d) are combined depends on the use of the number of ACs labeledwith a detectable label.

In another embodiment of the method of the first aspect of the presentinvention step (iv) comprises positively selecting (selecting) cellsbound to the 1^(st) AC, 1^(st) and 3^(rd) or 1^(st), 3^(rd) and 4^(th)AC. In another embodiment step (iv) comprises negatively selecting(excluding) cells bound to the 2^(nd) AC, the 2^(nd) and 5^(th) or the2^(nd), 5^(th) and 6^(th) AC. In another preferred embodiment the step(iv) comprises selecting cells bound to the 1^(st) AC, 1^(st) and 3^(rd)or 1^(st), 3^(rd) and 4^(th) AC and excluding cells bound to the 2^(nd)AC, the 2^(nd) and 5^(th) or the 2^(nd), 5^(th) and 6^(th) AC.

In another embodiment of the method of the first aspect of the presentinvention the detectable label is detected by flow cytometry analysis.In a preferred embodiment the detectable label is detected by FACSanalysis. In another preferred embodiment the detectable label isdetected by preparative sorting analysis.

In another embodiment of the method of the first aspect of the presentinvention the cells comprised in the population of step i) of the methodof the first aspect of the invention are T-cells and are phenotyped. Inanother embodiment the cells comprised in the population of step i) areB-cells and are phenotyped.

In another embodiment of the method of the first aspect of the presentinvention the phenotyping of T-cells comprises the determination of oneor more T-cell marker. T-cell marker are preferably selected from thegroup consisting of CD3, CD4, CD8, CD11a, CD14, CD19, CD25, CD27, CD28,CD44, CD45RA, CD45RO, CD57, CD62L, CD69, CD122, CD127, CD137 CD197(CCR7), IFNγ, IL-2, TNFα, IL7R and telomer length. In another preferredembodiment T-cell markers are CD45RA, CD45RO, CD197, CD25, CD27, CD57,CD95, CD127 and CD62L It is particularly preferred that CD69 and CD137are used for the phenotyping of T-cells. In another preferred embodimentthe phenotyping of B-cells comprises the determination of one or moreB-cell marker. B-cell marker are preferably selected from the groupconsisting of CD19, CD27, CD45R, CD21, CD40, CD20, CD38, and CD83.

In another embodiment of the method of the first aspect of the presentinvention the method further comprises the step of contacting the cellpopulation of step (i) with an irrelevant antigen complex (IAC)comprising an irrelevant protein antigen (IPA), wherein the amino acidsequence of the IPA when aligned with the amino acid sequence of the PAIis identical to the PAI at two amino acid positions or less and whereinthe IAC comprises a detectable label G that is detectably different fromthe detectable label A. Preferably, an irrelevant protein antigen is thegene product of a housekeeping gene. The housekeeping gene product isexpressed in all cells of an organism under normal andpathophysiological conditions which make it suitable to function as areference gene because it is usually not up or down regulated underdifferent or varying cell conditions. Generally, a natural immune cellpopulation should not comprise any immune cells binding to housekeepinggenes or peptides derived therefrom. Thus, the inclusion of an IPC inthe method of the invention allows the identification of T-cells thatnonspecifically bind to AC, which are also undesirable.

In another embodiment the amino acid sequence of at least one IP isselected by one or more of the following criteria: presentation of theIP on healthy tissue; the IP is derived from a HLA typed source; or thebinding to the respective HLA. It is preferred that the amino acidsequence of the IP or of at least one protein or peptide comprised inthe IPA has less than 50%, less than 40%, less than 30%, less than 29%,less than 28%, less than 27%, less than 26%, less than 25%, less than24%, less than 23%, less than 22%, less than 21%, less than 20%, lessthan 19%, less than 18%, less than 17%, less than 16%, less than 15%,less than 10%, less than 5% amino acid identity to the PAI. In anotherembodiment the IAC is a complex of a MHC-I or MHC-II and an IP. It ispreferred that the amino acid sequence of the IP when aligned with theamino acid sequence of the TP is identical to the TP at one or noneamino acid positions. Preferably, the IP is encoded by a housekeepinggene.

A second aspect of the invention further relates to a method fordetermining the sequence of a nucleic acid encoding an antigen-bindingprotein or an antigen-binding part thereof comprising the steps of:

-   (i) isolating the nucleic acid encoding the antigen-binding protein    or the antigen-binding part thereof from the cell selected in the    method of the first aspect of the invention; and-   (ii) determining the sequence of the nucleic acid.    In a preferred embodiment the nucleic acid is isolated from the    selected immune cell by methods well known in the art, e.g. organic    extraction, solid phase extraction, e.g. using a resin comprising a    styrene-divinylbenzene co-polymer containing iminodiacetic acid    groups. In another embodiment the isolated nucleic acid is either    DNA or RNA. In another embodiment it is preferred to amplify the    nucleic acid after isolation. Preferably, amplification is conducted    by polymerase chain reaction (PCR). More preferably the nucleic acid    is amplified in a rapid amplification of cDNA-ends with PCR (RACE    PCR). In another embodiment the synthesis of DNA from an RNA    template, via reverse transcription, produces complementary DNA    (cDNA). Reverse transcriptases (RTs) use an RNA template and a short    primer complementary to the 3′ end of the RNA to direct the    synthesis of the first strand cDNA, which can be used directly as a    template for the PCR. In another embodiment the sequence of the    isolated nucleic acid can be determined by known methods in the art,    for example next generation sequencing, e.g. Illumina (Solexa)    sequencing by simultaneously identifying DNA bases, as each base    emits a unique fluorescent signal, and adding them to a nucleic acid    chain, Roche 454 sequencing based on pyrosequencing, a technique    which detects pyrophosphate release, again using fluorescence, after    nucleotides are incorporated by polymerase to a new strand of DNA,    or ion torrent: Proton/PGM sequencing measuring the direct release    of protons from the incorporation of individual bases by DNA    polymerases.

A third aspect of the invention relates to a method for producing a cellexpressing a nucleic acid encoding an antigen-binding protein or anantigen-binding part thereof comprising the steps of:

-   (i) providing the nucleic acid sequence encoding the antigen-binding    protein or an antigen-binding part thereof from the cell selected in    the method of the first aspect of the invention;-   (ii) producing a nucleic acid vector comprising the nucleic acid    sequence provided in step (i) optionally under the control of an    expression control element; and-   (iii) introducing the nucleic acid vector of step (ii) into a host    cell.    In one embodiment the antigen-binding protein or an antigen binding    part thereof is cloned into a vector.

In one embodiment the antigen-binding protein is a TCR or an antigenbinding fragment thereof; a BCR or an antigen binding fragment thereofor an antibody or an antigen binding fragment thereof. In anotherembodiment, the antigen binding protein is a TCR or the part thereofcomprise at least the variable domains of the alpha and beta chain.Preferably, the sequence of the TCR or antigen binding part thereof isinserted into a suitable vector. In another embodiment the amino acidsequence of the TCR, BCR or antibody comprises six CDRs. In anotherembodiment two or three CDRs of the variable alpha and/or beta domain ofan identified TCR are inserted into the framework or another TCR orantibody. Preferably, the gene sequence of one, two or three CDRs of thevariable alpha domain of a TCR are cloned into a suitable vectorcomprising framework regions. The expression vector may either comprisenucleic acids encoding both the light or heavy chain or alpha and betachain (or the variable domains thereof)—soc-called “tandem type”—or theymay be encoded by nucleic acids comprised in separate vectors. It ispreferred that humanized antibody expression vectors of the tandem typeare used (shitara K et al. J Immunol Methods. 1994 Jan. 3;167(1-2):271-8). Examples of tandem type humanized antibody expressionvector include e.g. pKANTEX93 (WO 97/10354), and pEE18.

In another embodiment the vector of step (ii) is introduced into a hostcell. In one embodiment such recombinant host cells can be used for theproduction of at least one antigen binding protein of the invention orpart thereof. Preferably, the host cell is transformed, transduced ortransfected with a nucleic acid and/or a vector encoding the antigenbinding protein or antigen binding part thereof. Transduction ortransfection of host cells with nucleic acid encoding the antigenbinding protein or part of the antigen binding protein is conductedusing methods well known in the art, for example methods described inUS20190216852. In another embodiment the host cells comprising theantigen binding protein or antigen binding part thereof can be aeukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell can be acultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human. The host cell can be an adherent T-cell or asuspended cell, i.e., a cell that grows in suspension. For purposes ofproducing an antigen binding protein or part of the antigen bindingprotein, such as a recombinant TCR or fragment thereof, the host cell ispreferably a mammalian cell. Most preferably, the host cell is a humancell. While the host cell can be of any cell type, can originate fromany type of tissue, and can be of any developmental stage, the host cellpreferably is a peripheral blood leukocyte (PBL) or a peripheral bloodmononuclear cell (PBMC) or a B-cell. More preferably, the host cell is aT-cell. The T-cell can be any T-cell, such as a cultured T-cell, e.g., aprimary T-cell, or a T-cell from a cultured T-cell line, e.g., Jurkat,SupT1, etc., or a T-cell obtained from a mammal, preferably a T-cell orT-cell precursor from a human patient. If obtained from a mammal, theT-cell can be obtained from numerous sources, including but not limitedto blood, bone marrow, lymph node, the thymus, or other tissues orfluids. Preferably, the T-cell is a human T-cell. More preferably, theT-cell is a T-cell isolated from a human. The T-cell can be any type ofT-cell and can be of any developmental stage, including but not limitedto, CD4-positive and/or CD8-positive, CD4-positive helper T-cells, e.g.,Th1 and Th2 cells, CD8-positive T-cells (e.g., cytotoxic T-cells), tumorinfiltrating cells (TILs), memory T-cells, naive T-cells. Preferably,the T-cell is a CD8-positive T-cell or a CD4-positive T-cell. In anotherembodiment the host cell may be any cell for recombinant expression.Preferably, the host cell is a Chinese hamster ovary (CHO) cell.

A fourth aspect of the invention relates to a method for treating asubject in need thereof comprising the steps of:

-   (i) providing a cell population of the subject comprising immune    cells;-   (ii) contacting the cell population of step (i) with a first antigen    complex (1^(st) AC) comprising a PAI and a detectable label A or    with the PAI comprising a detectable label A;-   (iii) contacting the cell population of step (i) with at least a    second antigen complex (2^(nd) AC) comprising a SPA, wherein the    amino acid sequence of the SPA differs by at least 1 amino acid from    the amino acid sequence of the PAI and wherein the 2^(nd) AC    comprises a detectable label B; and-   (iv) selecting at least one cell that specifically binds to the    1^(st) AC,    -   wherein the detectable label A and the detectable label B are        detectably different from each other-   (v) increasing the number of the at least one selected cell by    cultivation; and-   (vi) reintroducing the cultivated cells into the subject.

This approach is an ACT approach. Preferably, the selected cells aretransferred into the subject after being genetically engineered andfunctionally improved. Preferably, the cells are originated from thesubject to which they are transferred to after being geneticallyengineered, i.e. donor of the cells and receptor of engineered cells isidentical. The subject is a subject in need thereof as defined hereinabove. Preferably, the subject in need thereof suffers or is at risk ofsuffering from a disease selected from the group consisting of immunediseases or neoplastic diseases, a disease caused by a virus or adisease caused by bacteria. It is preferred that the neoplastic diseaseis cancer. It is preferred that the disease caused by a virus is HIV. Itis preferred that the disease cause by a bacterium is tuberculosis.

A fifth aspect of the invention relates to a method for selecting animmune cell expressing on its surface an antigen-binding proteinspecifically binding to a protein antigen of interest (PAI) comprisingthe following steps:

-   (i) providing a cell population comprising immune cells;-   (ii) contacting the cell population of step (i) with a first antigen    complex (1^(st) AC) comprising the PAI and a detectable label A or    with the PAI comprising a detectable label A;-   (iii) contacting the cell population of step (i) with at least a    second antigen complex (2^(nd) AC) comprising an irrelevant protein    antigen (IPA), wherein the amino acid sequence of the IPA when    aligned with the amino acid sequence of the PAI is identical to the    PAI at two amino acids positions or less and wherein the IAC    comprises a detectable label G; or with the IPA and a detectable    label G; and-   (iv) selecting at least one cell that specifically binds to the    1^(st) AC,    wherein the detectable label A and the detectable label G are    detectably different from each other. The selection process    according to the fifth aspect of the invention is carried out as    outlined above for the first aspect of the invention.

EXAMPLES Example 1: Direct Sorting of Target-Peptide Specific T Cellswith and without Prior Target-Specific Expansion

FIGS. 2 and 3 show a comparison of two different approaches which leadto sorting of target-specific T cells while sparing cross-reactive Tcells which recognize target and similar peptides. Method 1 (FIG. 2)does not require prior amplification of target-specific T cells. PBMCsare isolated and enriched for T cell populations by magnetic beadseparation. The T cell population is stained withfluorochrome-conjugated target-peptide and similar-peptide tetramers.Subsequently, those cells can be further enriched for target-specific Tcells by using magnetic bead separation targeting one of thefluorochrome-conjugates of the target-tetramers. The targetpeptide-specific T cell population is then stained for surface markerssuch as CD4 and CD8 as well as viability markers to exclude dead cells.By using flow cytometric sorting approaches the target-specific T cellscan be sorted for desired surface marker expression while sparingtarget+similar peptide-specific T cells as shown in FIG. 2. Method 2(FIG. 3) utilizes stimulation with target-peptide HLA-coated artificialantigen-presenting cells to amplify low frequency target-specific Tcells. Here, enriched CD8 T cells are cultivated in individual vesselsto allow for the growth of mono- or oligoclonal target-peptide specificT cell populations. After repeated stimulation with artificialantigen-presenting cells the individual mono- or oligoclonal populationsare stained with surface markers as well as target- and similar-peptidetetramers (target and similar peptide tetramers are labelled with 2distinct fluorochromes each in a 2D staining approach) which allows fordistinction of target-specific and cross-reactive mono- or oligoclonal Tcell populations as shown in FIG. 3.

Example 2: Functional Assessment of T Cell Receptors Derived from TCells Sorted with Target-Peptide Multimers Only

To assess functionality and specificity of TCRs identified by sortingwith target multimers, T cell receptor mRNA is generated by in vitrotranscription and subsequently used to transfect CD8 positive T cells ofhealthy donors by electroporation. Eighteen hours after electroporation20,000 transfected T cells are then co-incubated with T2 cells loadedeither with target peptide, different target-sequence similar peptides,an irrelevant peptide or unloaded T2 cells at a 1:1 ratio. Supernatantsare harvested 24 h after start of co-culture and analyzed for secretedIFN-γ by ELISA-technique. Cytokine secretion demonstrates antigenrecognition and activity of the respective T cells as illustrated inFIG. 4. Whereas all TCRs in FIGS. 4A, B and C recognize the target(positive control), TCRs in FIG. 4A and FIG. 4B are also cross-reactivetowards target-sequence similar peptides expressed on normal tissue andare thus excluded from further analysis, only “clean” TCRs (FIG. 4C) areworth to be selected for further characterization. (“Target”=targetpeptide; TP; “SIM 1-10”=target similar peptides; TSPs 1-10).

Peptides Used in this Example:TP and SIM 1-SIM 10 are all 9mers.

-   -   TP and SIM 1 differ in amino acid position 2, 5, 8 and 9 wherein        SIM 1 has an isoleucine residue at position 2, a threonine        residue at position 5, a leucine residue at position 8 and a        valine residue at position 9.    -   TP and SIM 2 differ in position 3, 4 and 7, wherein SIM 2 has an        isoleucine residue at position 3, a glutamic acid residue at        position 4 and a glutamine residue at position 7.    -   TP and SIM 3 differ in position 2, 7, 8 and 9, wherein SIM 3 has        an isoleucine residue at position 3, a glutamic acid residue at        position 7 and 8 and an isoleucine residue at position 9.    -   TP and SIM 4 differ in position 4, 5 and 8, wherein SIM 4 has a        lysine residue at position 4, an asparagine residue at position        5 and a tyrosine residue at position 8.    -   TP and SIM 5 differ in position 4, 7 and 8, wherein SIM 5 has an        asparagine residue at position 4, a proline residue at position        7 and a tyrosine residue at position 8.    -   TP and SIM 6 differ in position 6, 7 and 8, wherein SIM 6 has        valine residue at position 6 and a leucine residue at position 7        and 8.    -   TP and SIM 7 differ in position 5, 6 and 8, wherein SIM 7 has        lysine residue at position 5, a glutamine residue at position 6        and a methionine residue at position 8.    -   TP and SIM 8 differ in position 3, 5, 7 and 9, wherein SIM 8 has        serine residue at position 3, a glutamic acid residue at        position 5 and a valine residue at position 7 and 9.    -   TP and SIM 9 differ in position 2, 4, 5 and 9, wherein SIM 9 has        valine residue at position 2, a glycine residue at position 4,        an alanine residue at position 5 and a valine residue at        position 9.    -   TP and SIM 10 differ in position 1, 4 and 6, wherein SIM 10 has        valine residue at position 1, a histidine residue at position 4        and a glutamine residue at position 9.    -   TP and control peptide differ in positions 4-9.

Example 3: Functional Assessment of T Cell Receptors Derived fromTarget-Peptide as Well as Target- and Similar-Peptide Specific T Cells

To this end, T cell receptor mRNA is generated using in vitrotranscription and subsequently used to transfect NFAT-luciferase Jurkatcells by electroporation. The transfected Jurkat cells start to expressthe newly introduced TCRs transiently on their surface. Eighteen hoursafter electroporation 50,000 Jurkat cells are then co-incubated with T2cells at a 1:1 ratio loaded either with a target peptide or the similarpeptides which are used for sorting, as well as a control peptide or nopeptide. Upon specific binding of the TCR to its cognate peptide-HLA,signaling leads to NFAT activation which in turn leads to expression ofluciferase. After overnight incubation luciferase substrate is added anda luminescence signal can be detected when the T cell is activated. FIG.5 shows that TCRs derived from target-tetramer binding T cells lead tofunctional activation when stimulated with target-peptide loaded T2cells. (“Target”=target peptide; TP; “SIM 1-3”=target similar peptides;TSPs 1-3).

Peptides Used in this Example:TP and SIM 1-SIM 3 are all 9mers.

-   -   TP and SIM 1 differ in amino acid position 4, 6 and 7 wherein        SIM 1 has a glutamic acid residue at position 4, a leucine        residue at position 6 and an isoleucine residue at position 7.    -   TP and SIM 2 differ in position 2, 7 and 8, wherein SIM 2 has a        methionine residue at position 2, a glutamic acid residue at        position 7 and lysine residue at position 8.    -   TP and SIM 3 differ in position 1, 5 and 6, wherein SIM 3 has a        phenylalanine residue at position 1, a glycine residue at        position 5 and a serine residue at position 6.    -   TP and control peptide differ in positions 1 and 4-9.

Overall SIM 1 has a similarity to TP of 77%, SIM 2 has a similarity toTP of 77% and SIM 3 has a similarity to TP of 75% using BLASTP, BLOSUM62scoring matrix, a word length of 3, and expectation (E) of 10.

Example 4: Relevant and Irrelevant Target Similar Peptides for a GivenTarget Peptide

The relevance of a peptide as TSP to a given TP is determined mainly byits similarity to the TP, and can additionally be guided by itsfrequency of presentation as well as and quantitative presentation level(copy numbers per cell (CpC)) on primary normal tissues. The higher thesimilarity to the TP and the higher the presentation frequency and CpCon normal tissues, the higher the relevance of a TSP. Table 6 showsexample sequences of a TP, two corresponding TSPs as well as an IP. Perpeptide, the number of identical amino acids (aa) to the TP, thesimilarity based on the pmbec positional scoring matrix in comparison tothe TP sequence and the CpC range on normal tissues is depicted. FIGS.6, 7 and 8 additionally shows the peptide presentation profiles of thetwo TSPs as well as the IP. TSP (TSP1) has 4 identical amino acids tothe TP but a higher overall similarity to the target as compared to TSP2which has 5 identical amino acids in comparison to the target peptide.Both TSPs are considered relevant based on their similarity to the TPand their presentation on normal tissues (FIGS. 6 and 7). The depictedIP shows an even higher presentation frequency on normal tissues (FIG.8) and is in general also presented at a higher copy number per cell.The sequence similarity as well as the number of identical amino acidsis however rather low (17% similarity and 0 identical amino acids).

TABLE 6 Similarity Number of to TP CpC range Amino acid identical (PMBECnormal equence aa to TP Score) tissue TP VLLHHQIGL 9 100%  n.a.(SEQ ID NO: 164) TSP1 ALMYHTITL 4 63% 5-60 (SEQ ID NO: 165) TSP2LLLAHIIAL 5 55% 15-35  (SEQ ID NO: 166) IP AIVDKVPSV 0 17% 55-600(SEQ ID NO: 167)

1. A method for selecting a cell or a virus expressing on its surface anantigen-binding protein specifically and/or selectively binding to aprotein antigen of interest (PAI) comprising the following steps: (i)providing a cell population or a virus population; (ii) contacting thecell population or the virus population of step (i) with a first antigencomplex (1^(st) AC) comprising the PAI and a detectable label A or withthe PAI comprising a detectable label A; (iii) contacting the cellpopulation or the virus population of step (i) with at least a secondantigen complex (2^(nd) AC) comprising a similar protein antigen (SPA),wherein the amino acid sequence of the SPA differs by at least 1 aminoacid from the amino acid sequence of the PAI and wherein the 2^(nd) ACcomprises a detectable label B; or with the SPA and a detectable labelB; and (iv) selecting at least one cell or virus that specificallyand/or selectively binds to the 1^(st) AC, wherein the detectable labelA and the detectable label B are detectably different from each other.2. The method according to claim 1, wherein (i) the selected cell is animmune cell, preferably a T-cell, preferably a CD4 or CD8 T-cell; or aB-cell; or a mammalian or yeast cell expressing a heterologous antigenbinding protein; or (ii) the selected virus is a bacteriophage.
 3. Themethod according to claim 1, wherein the antigen-binding protein isselected from the group comprising a T-cell receptor (TCR) or antigenbinding fragments thereof, a B-cell receptor (BCR) or antigen bindingfragments thereof, and a chimeric antigen receptor (CAR) or antigenbinding fragments thereof.
 4. The method according to claim 1, wherein(a) the cell population comprises: (i) immune cells preferablytumor-infiltrating lymphocytes (TILs), T cell receptor libraries,peripheral blood of healthy subjects, peripheral blood of diseasedsubjects or an immune cell enriched fraction thereof; or (ii) eukaryoticcells, preferably mammalian cells or yeast cells expressing a library ofheterologous antigen binding proteins; or (b) the virus populationcomprises viruses expressing a library of heterologous antigen bindingproteins.
 5. The method according to claim 4, wherein the immune cellenriched fraction is enriched in stem cells; T-cells, preferably CD8T-cells or CD4 T-cells; B-cells; plasma cell.
 6. The method according toclaim 1, wherein the protein antigen of interest (PAI) is a tumorassociated antigen (TAA), a viral protein or a bacterial protein.
 7. Themethod according to claim 4, wherein the diseased subject suffers from adisease selected from the group consisting of immune diseases,neoplastic diseases, a disease caused by a virus or a disease caused bybacteria.
 8. The method according to claim 4, wherein the immune cellenriched fraction is selected by detectably labeling one or more immunecell specific surface marker.
 9. The method according to claim 1,comprising the further step of incubating the cell population in thepresence of growth and/or differentiation factors, preferably selectedfrom the group consisting of cytokines.
 10. The method according toclaim 1, wherein the AC is an antigen-presenting cell, or a complexcomprising a particle, the PAI and the detectable label A or the SPA andthe detectable label B.
 11. The method according to claim 1, comprisingone or more of the following further steps: (a) contacting the cellpopulation of step (i) with a third antigen complex (3^(rd) AC)comprising the PAI and a detectable label C that is detectably differentfrom one or more or all of the other detectable labels of the other ACscontacted with the cell population, preferably detectably different fromat least the detectable label A, preferably from at least the detectablelabel A and a detectable label D, if a detectable label D is present;and/or (b) contacting the cell population of step (i) with a fourthantigen complex (4^(th) AC) comprising the PAI and a detectable label Dthat is detectably different from one or more or all of the otherdetectable labels of the other ACs contacted with the cell population,preferably detectably different from at least the detectable label Aand, preferably from at least the detectable label A and the detectablelabel C; and/or (c) contacting the cell population of step (i) with afifth antigen complex (5^(th) AC) comprising the SPA and a detectablelabel E that is detectably different from one or more or all of theother detectable labels of the other ACs contacted with the cellpopulation, preferably detectably different from at least the detectablelabel B and, preferably from at least the detectable label B and adetectable label F, if a detectable label F is present; and/or (d)contacting the cell population of step (i) with a sixth antigen complex(6^(th) AC) comprising the SPA and a detectable label F that isdetectably different from one or more or all of the other detectablelabels of the other ACs contacted with the cell population, preferablydetectably different from at least the detectable label B and,preferably from at least the detectable label B and the detectable labelE; and/or (e) contacting the cell population of step (i) with one ormore further antigen complexes (AC) wherein each comprises a SPA thatdiffers in at least one amino acid sequence from the amino acid sequenceof the SPA of the 2^(nd) AC, and wherein each further AC comprises oneor more labels, wherein the one or more label is the same to ordetectably different from the one or more labels of the 2^(nd) AC. 12.The method according to claim 1, wherein (i) the 1^(st) AC comprises atleast one further detectable label and the 2^(nd) AC comprises at leastone further detectable label, which are either the same or different;and/or (ii) the one or more further AC comprises at least one furtherdetectable label; wherein the at least one further label is selected insuch that it allows to distinguish the 1^(st) AC from the 2^(nd) AC andthe one or more further ACs.
 13. The method according to claim 1,wherein the detectable labels are independently selected from afluorescent label, preferably selected from the group consisting ofxanthens, acridines, oxazines, cyanines, styryl dyes, coumarines,porphines, metal-ligand-complexes, fluorescent proteins, nanocrystals,perylenes and phtalocyanines.
 14. The method according to claim 1,wherein the 1^(st) AC is a complex of a MHC-I or MHC-II and the PAI, andwherein the PAI is a target peptide (TP), preferably a tumor-specifictarget peptide and/or the 2^(nd) AC is a complex of a MHC-I or MHC-IIand the SPA, and wherein the SPA is a target similar peptide (TSP) andwherein the TSP differs by at least 1 amino acid from the amino acidsequence of the TP.
 15. The method according to claim 14, wherein theamino acid sequence of the at least one TSP is selected by one or moreof the following criteria: (a) presentation of the TSP on healthytissue; (b) derived from HLA typed source; and (c) binding to therespective HLA.
 16. The method according to claim 14, wherein the aminoacid sequence of the TSP has a length of 8 to 16 amino acids andwherein: (1) the amino acid sequence of the TSP differs from the aminoacid sequence of the TP X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈ (i) at position X₁, X₂and X₃, and wherein position X₄ to X₈ are identical or similar to theTP; (ii) at position X₄, X₅ and X₆, and wherein positions X₁ to X₃ andX₇ and X₉ are identical or similar to the TP; or (iii) at position X₇and X₈, and wherein position X₁ to X₆ are identical or similar to theTP; or if the TP has a length of 8 amino acids; or (2) the amino acidsequence of the TSP differs from the amino acid sequence of the TPX₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉ (i) at position X₁, X₂ and X₃, and whereinposition X₄ to X₉ are identical or similar to the TP; (ii) at positionX₄, X₅ and X₆, and wherein position X₁ to X₃ and positions X₇ to X₉ areidentical or similar to the TP; or (iii) at position X₄, X₅, X₆ and X₇,and wherein position X₁ to X₃ and positions X₈ to X₉ are identical orsimilar to the TP; or (iv) at position X₇ X₈ and X₉, and whereinposition X₁ to X₆ are identical or similar to the TP; or if the TP has alength of 8-9 amino acids; or (3) the amino acid sequence of the TSPdiffers from the amino acid sequence of the TPX₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀ (i) at position X₁, X₂ and X₃, whereinposition X₄ to X₁₀ are identical or similar to the TP; (ii) at positionX₄, X₅, X₆ and X₇, wherein position X₁ to X₃ and positions X₈ to X₁₀ areidentical or similar to the TP; or (iii) at position X₄, X₅ and X₆, andwherein position X₁ to X₃ and positions X₇ to X₁₀ are identical orsimilar to the TP; or (iv) at position X₈, X₉ and X₁₀, wherein positionX₁ to X₇ are identical or similar to the TP; or if the TP has a lengthof 8-10 amino acids; or (4) the amino acid sequence of the TSP differsfrom the amino acid sequence of the TP X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀X₁₁(i) at position X₁, X₂ and X₃, wherein position X₄ to X₁₁ are identicalor similar to the TP; (ii) at position X₄, X₅, X₆ and X₇, whereinposition X₁ to X₃ and positions X₈ to X₁₁ are identical or similar tothe TP; or (iii) at position X₄, X₅ and X₆, and wherein position X₁ toX₃ and positions X₇ to X₁₁ are identical or similar to the TP; or (iv)at position X₈, X₉, X₁₀ and X₁₁, wherein position X₁ to X₇ are identicalor similar to the TP; or (v) at position X₉, X₁₀ and X₁₁, whereinposition X₁ to X₈ are identical or similar to the TP; if the TP has alength of 8-11 amino acids; or (5) the amino acid sequence of the TSPdiffers from the amino acid sequence of the TPX₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀X₁₁ X₁₂ (i) at position X₁, X₂ and X₃,wherein position X₄ to X₁₂ are identical or similar to the TP; (ii) atposition X₄, X₅, X₆ and X₇, wherein position X₁ to X₃ and positions X₈to X₁₂ are identical or similar to the TP; or (iii) at position X₄, X₅and X₆, and wherein position X₁ to X₃ and positions X₇ to X₁₂ areidentical or similar to the TP; or (iv) at position X₈, X₉, X₁₀, X₁₁ andX₁₂, wherein position X₁ to X₇ are identical or similar to the TP; or(v) at position X₉, X₁₀, X₁₁ and X₁₂, wherein position X₁ to X₈ areidentical or similar to the TP; if the TP has a length of 8-12 aminoacids. 17.-18. (canceled)
 19. The method according to claim 1, whereinthe cell population of step (i) is contacted with not more than 10antigen complexes (AC) each comprising a different similar proteinantigen (SPA), not more than nine different SPAs, not more than eightdifferent SPAs, not more than seven different SPAs, not more than sixdifferent SPAs, not more than five different SPAs, not more than fourdifferent SPAs, not more than three different SPAs, not more than twodifferent SPAs, or not more than one SPA, is used.
 20. The methodaccording to claim 19, wherein the SPA is a TSP.
 21. The methodaccording to claim 20, wherein the number of different TSPs is between1-10; between 2-8; between 3-5 or between 1-3, preferably three TSPs areused.
 22. (canceled)
 23. The method according to claim 1, wherein step(iv) comprises: a) positively selecting (selecting) cells bound to the1^(st) AC, 1^(st) and 3^(rd) or 1^(st), 3^(rd) and 4^(th) AC; and/or b)negatively selecting (excluding) cells bound to the 2^(nd) AC, the2^(nd) and 5^(th) or the 2^(nd), 5^(th) and 6^(th) AC. 24.-39.(canceled)